Standardizing terms, definitions and concepts for describing and interpreting unwanted immunogenicity of biopharmaceuticals: recommendations of the Innovative Medicines Initiative ABIRISK consortium.

Biopharmaceuticals (BPs) represent a rapidly growing class of approved and investigational drug therapies that is contributing significantly to advancing treatment in multiple disease areas, including inflammatory and autoimmune diseases, genetic deficiencies and cancer. Unfortunately, unwanted immunogenic responses to BPs, in particular those affecting clinical safety or efficacy, remain among the most common negative effects associated with this important class of drugs. To manage and reduce risk of unwanted immunogenicity, diverse communities of clinicians, pharmaceutical industry and academic scientists are involved in: interpretation and management of clinical and biological outcomes of BP immunogenicity, improvement of methods for describing, predicting and mitigating immunogenicity risk and elucidation of underlying causes. Collaboration and alignment of efforts across these communities is made difficult due to lack of agreement on concepts, practices and standardized terms and definitions related to immunogenicity. The Innovative Medicines Initiative (IMI; www.imi-europe.org), ABIRISK consortium [Anti-Biopharmaceutical (BP) Immunization Prediction and Clinical Relevance to Reduce the Risk; www.abirisk.eu] was formed by leading clinicians, academic scientists and EFPIA (European Federation of Pharmaceutical Industries and Associations) members to elucidate underlying causes, improve methods for immunogenicity prediction and mitigation and establish common definitions around terms and concepts related to immunogenicity. These efforts are expected to facilitate broader collaborations and lead to new guidelines for managing immunogenicity. To support alignment, an overview of concepts behind the set of key terms and definitions adopted to date by ABIRISK is provided herein along with a link to access and download the ABIRISK terms and definitions and provide comments (http://www.abirisk.eu/index_t_and_d.asp).

Retinoic acid maintains differentiated cell morphology and functions in long-term cultured porcine hepatocytes: obtaining functional cells for prolonged treatments with bioartificial liver.

Porcine hepatocytes show several immunological characteristics and enzymatic activities of human liver, representing an ideal xenogenic source of cells as biological component of bioartificial liver (BAL). Isolated hepatocytes rapidly lose their specific metabolic activities and their typical morphology when cultured in the presence of serum. Since in BAL porcine hepatocytes are perfused by the patient's plasma, procedures able to minimize de-differentiation of cells could be useful for long-term treatment of acute liver failure (ALF). In this work we found that, in the presence of micromolar concentration of All trans-retinoic acid (ATRA), porcine parenchymal liver cells undergo to a lower extent the de-differentiating effects of long-term culture in the presence of serum. The evaluation of lidocaine metabolism showed that ATRA-treated cells retain specific hepatocyte function for a significantly longer time when compared to control hepatocytes. A tyrosine phosphorylation of PLC-gamma1 was observed in concomitance with the ATRA-induced maximal functional activity. An increased expression of PLC-beta3 and PKC-alpha and -beta2 was also evidentiated at the longer time points explored, when the effects of ATRA in preservation of the differentiated morphology were maximal. These results provide the first evidence that ATRA plays a differentiating role in adult porcine hepatocytes cultured under de-differentiating conditions. The administration of ATRA to isolated parenchymal cells from pig liver may provide functional hepatocytes for prolonged treatment with BAL.

A macrophage invasion mechanism for mycobacteria implicating the extracellular domain of CD43.

We studied the role of CD43 (leukosialin/sialophorin), the negatively charged sialoglycoprotein of leukocytes, in the binding of mycobacteria to host cells. CD43-transfected HeLa cells bound Mycobacterium avium, but not Salmonella typhimurium or Shigella flexneri. Quantitative bacteriology showed that macrophages (M(phi)) from wild-type mice (CD43(+/+)) bound M. avium, Mycobacterium bovis (bacillus Calmette-Guérin), and Mycobacterium tuberculosis (strain H37Rv), whereas M(phi) from CD43 knockout mice (CD43(-/)-) did not. Fluorescence microscopy demonstrated that the associated M. avium had been ingested by the CD43(+/+) M(phi). The inability of CD43(-/)- M(phi) to bind M. avium could be restored by addition of galactoglycoprotein (Galgp), the extracellular mucin portion of CD43. The effect of Galgp is not due to opsonization of the bacteria, but required its interaction with the M(phi) other mucins had no effect. CD43 expression by the M(phi) was also required for optimal induction by M. avium of tumor necrosis factor (TNF)-alpha production, which likewise could be reconstituted by Galgp. In contrast, interleukin (IL)-10 production by M. avium-infected M(phi) was CD43 independent, demonstrating discordant regulation of TNF-alpha and IL-10. These findings describe a novel role of CD43 in promoting stable interaction of mycobacteria with receptors on the M(phi) enabling the cells to respond specifically with TNF-alpha production.

Macrophage apoptosis in mycobacterial infections.

Mycobacterial diseases are a major public health concern. In the case of tuberculosis, the problem has been acerbated due to the emergence of drug-resistant strains of Mycobacterium tuberculosis, and Mycobacterium avium is the major opportunistic pathogen in HIV-1 infection in the United States. M. tuberculosis and M. avium replicate in human macrophages and induce apoptosis. Incubation of freshly added uninfected autologous macrophages with apoptotic M. avium-infected macrophages results in 90% inhibition of bacterial growth. Apoptosis also prevents the release of intracellular components and the spread of mycobacterial infection by sequestering the pathogens within apoptotic bodies. Consistent with the model that host cell apoptosis is a defense mechanism against mycobacteria is the finding that the virulent M. tuberculosis strain H37Rv induces substantially less macrophage apoptosis than the attenuated strain H37Ra. Evasion of apoptosis by this pathogen is achieved by enhanced release of sTNFR2 by H37Rv-infected macrophages and subsequent formation of inactive TNF-alpha-TNFR2 complexes. These observations contribute to the hypothesis that apoptosis of the host macrophage is an important defense mechanism in mycobacterial infections, which prevents the spread of the infection.

Daptomycin: a novel agent for Gram-positive infections.

The alarming increase in the incidence of Gram-positive infections, including those caused by resistant bacteria, has sparked renewed interest in novel antibiotics. One such agent is daptomycin, a novel lipopeptide antibiotic with proven bactericidal activity in vitro against all clinically relevant Gram-positive bacteria. These include resistant pathogens, such as vancomycin-resistant enterococci (VRE), methicillin-resistant Staphylococcus aureus (MRSA), glycopeptide intermediately susceptible Staphylococcus aureus (GISA), coagulase-negative staphylococci (CNS) and penicillin-resistant Streptococcus pneumoniae (PRSP), for which there are very few therapeutic alternatives. Daptomycin provides rapid, concentration-dependent killing and a relatively prolonged concentration-dependent post-antibiotic effect in vitro. Spontaneous acquisition of resistance to daptomycin occurs rarely. Daptomycin exhibits linear pharmacokinetics, minimal accumulation with once-daily dosing, and low plasma clearance and volume of distribution. Phase II clinical trials indicate that daptomycin at doses of 2 mg/kg q24 h and 3 mg/kg q12 h is efficacious against skin and soft tissue infections and bacteremia, respectively. In addition, results in endocarditis suggested potential efficacy with higher doses. On the basis of clinical trials to date, it appears that daptomycin has an excellent safety profile, with the incidence and nature of serious adverse events comparable to those observed with conventional therapy. Adverse events associated with other classes of antimicrobials (nephrotoxicity, local irritation, ototoxicity, hypersensitivity, and gastrointestinal effects) were uncommon with daptomycin. Minimal skeletal muscle toxicity was seen at only the highest dose tested (4 mg/kg q12 h), predicted by elevations in serum creatinine phosphokinase, and readily reversible upon discontinuation of treatment. There were no signs of toxicity in cardiac or smooth muscle. Phase II and III clinical trials are underway to evaluate daptomycin for the treatment of Gram-positive bacteremia and complicated skin and soft tissue infections, respectively. Daptomycin holds promise as a rapidly acting and highly effective antibiotic for Gram-positive infections.

Regulation of human cytotoxic T lymphocytes development by the synergistic effect of IL-7 and sCD23.

The present study was undertaken to investigate the interaction of IL-7 and sCD23 on human peripheral blood T cell activation and CTL differentiation. Purified T lymphocytes were stimulated with mitogen plus IL-2 and subcultured for 7 days with IL-7 and/or sCD23. The combination of IL-7 and sCD23 synergistically enhanced the proliferation of both CD4+ and CD8+. T cells. CD8+ T cells, however, were usually more responsive to IL-7 and sCD23. This synergy was observed on both subsets of T cells. Furthermore, these cytokines synergistically augment the CTL activity of CD8+ T cells in both mitogen- and antigen-activated T cells. MAbs anti-IL-2 or anti-IL-2R (CD25) and anti-IL-12 had no effect on T cell proliferation and CD8+ cytotoxic activity induced by IL-7 and sCD23. We analyzed the effect on IFN-gamma induction by CD8+ T cells and found that IL-7 alone was incapable of inducing detectable levels of IFN-gamma production, but together with sCD23 it enhanced the production of IFN-gamma. We also found that IFN-gamma was not required for enhanced CTL activity of CD8+ T cells, because rabbit anti-IFN-gamma did not block the synergistic effects of either cytokine. The data demonstrate that the synergistic stimulatory activity of IL-7 and sCD23 may be of significance in the human CTL development and provide an alternative mechanism of stimulating T cells for use in immunotherapy.

Modulation of TCR usage in HIV-1 infection is regulated by IL-7 and sCD23.

The purpose of this study is to elucidate the effect of interleukin-7 (IL-7) and soluble CD23 (sCD23) on Phorbol12 Myristate13 Acetate (PMA) activated CD4+ TCR alpha beta+ cells of HIV-1 infected subjects. CD23 and IL-7R were detectable on activated CD4+ T cells of these subjects by FACS. Addition on IL-7 (1000 U/ml), at the onset of cultures, resulted in a significant increase of CD23 expression. We also demonstrated that T cells proliferation and CD23 expression in the presence of exogenous IL-7 occur in an IL-2 independent manner. Addition of IL-7 and sCD23 to activated CD4+ cells of HIV-1 infected subjects induced a proliferative response of TCR alpha beta cells. In contrast, addition of either sCD23 or IL-7 to activated CD4+ T cells did not result in an increase of TCR alpha beta expression. The data provide direct evidence that sCD23 in combination with IL-7 induce proliferation of activated CD4+ T cells of HIV-1 infected subjects to augment TCR alpha beta expression. These results support the possibility that IL-7 plus sCD23 might play an important role in the modulation of TCR alpha beta expression in HIV infection.

Interleukin 7 and sCD23 synergize in the induction of human T cell activation in HIV-1-infected subjects.

The role played by CD23 in retroviral infections is still unclear. The synergistic effects of interleukin 7 (IL-7) and sCD23 on T cell proliferation and the generation of HIV-1-specific cytotoxic T lymphocytes (CTL) in mitogen- and antigen-stimulated systems were examined. Addition of IL-7 and sCD23 at the onset of culture resulted in a marked augmentation of T cell proliferation and cytotoxic activity. Studies of CTL development in purified mitogen CD8+ T cells demonstrated that IL-7 and sCD23 could act directly on the CD8+ lymphocyte subset to augment cytotoxicity. The data demonstrate that IL-7 and sCD23 synergistically augmented CTL activity independently of IL-2 and IL-12. We analyzed the effects on IFN-gamma production by CD8+ T cells and found that IL-7 alone did not induce detectable levels of IFN-gamma production, but together with sCD23, it synergistically enhanced the production of IFN-gamma. We also found that IFN-gamma appeared not to be required for the enhanced CD8+ CTL activity because rabbit anti-IFN-gamma antibody did not block the synergistic effects of IL-7 and sCD23. These results indicate that IL-7 and sCD23 can exert major upregulatory effects on human CTL development and suggest that these effects are both proliferative and differentiative.

Changes of nuclear PI-PLC gamma1 during rat liver regeneration.

We have previously demonstrated that rat liver nuclei contain PI-PLC beta1 and gamma1 in the inner nuclear matrix and lamina associated with specific phosphodiesterase activity (Bertagnolo et al., 1995, Cell Signall. 7, 669-678). Since compensatory hepatic growth is an informative and well characterized model for natural cell proliferation, the presence of specific PI-PLC isoforms and their activity as well as PIP2 recovery were studied at various regenerating times, ranging from 3 to 22 h after partial hepatectomy. Three PI-PLC isoforms (beta1, gamma1, delta1) were examined in control and regenerating liver cells by using specific antibodies. By means of in situ immunocytochemistry and confocal microscopy, PI-PLC beta1 was found mainly in the nucleoplasm and this pattern was not modified after hepatectomy. On the contrary, the nuclear gamma1 isoform showed a marked decrease at 3 and 16 h after hepatectomy, but a clear increase at 22 h covering with bright intensity the whole nucleus. The PI-PLC delta1 isoform, which is exclusively cytoplasmic, was not altered during rat liver regeneration. By western blotting analysis on whole cell homogenates, none of the PI-PLC isozymes under study showed proliferation-linked modification. However, analyses of isolated nuclei identified changes in the nucleus associated PI-PLC gamma1 that paralleled the in situ observation whereas the beta1 isoform was unmodified at all the times examined. Nuclear phosphodiesterase activity on PIP2 was lower at 3 and 16 h, in comparison with sham operated rats, increased at 6 h and reached the highest value after 22 h. Consistently, the recovery of PIP2, obtained in conditions that optimise PIP-kinase activity, showed a marked decrease at 3 h and an increase up to 16 h of liver regeneration, followed by a further decrease at 22 h. These data are consistent with a close relationship between cell proliferation and the nuclear inositide cycle, depending, in rat liver, predominantly on the modulation of the gamma1 isoform of PI-PLC.

Interleukin-7 modulates CD23 and HLA-DR expression on CD4+ T cells and promotes a Th-2 type citokine profile.

The expression of CD23 on PHA-activated human PBT (peripheral blood T) cells of healthy donors was investigated. It appears that CD23 is expressed solely on activated CD4+ T cells. Cytofluorotometric analysis revealed that 6% of PHA-activated CD4+ T cells expressed CD23, while unstimulated CD4+ T cells express no detectable CD23. The addition of IL-7 (1000 U/ml) to activated CD4+ T cells resulted in a marked augmentation of CD23 expression (29%). CD23 expression was blocked by M20 and M26 mAbs, but no reduction was detected by anti-IL-2R (CD25) mAb. This suggests that IL-7 has a specific regulatory effect on CD23 expression independent of IL-2. Northern Blot analysis showed a marked increase of CD23 mRNA detected in PHA-activated CD4+ T cells plus IL-7. IL-7 was also able to upregulate the expression of HLA-DR on activated CD4+ T cells. Optimal HLA-DR and CD23 induction by IL-7 occurred at 48 and 72 h of culture. The addition of CHX revealed that the induction of CD23 and HLA-DR by IL-7 required intact protein synthesis. Furthermore, PHA activated CD4+ T cells cultured in the presence of IL-7 are polarized to a Th-2 pattern of cytokine production.

Programmed cell death of Mycobacterium avium serovar 4-infected human macrophages prevents the mycobacteria from spreading and induces mycobacterial growth inhibition by freshly added, uninfected macrophages.

Mycobacterium avium, an opportunistic pathogen in AIDS patients, replicates in human macrophages (Mphi) and induces programmed cell death (PCD). In this study we examine the effect of freshly added, uninfected Mphi on M. avium growth in apoptotic Mphi cultures. Incubation of uninfected autologous Mphi with apoptotic Mphi infected with M. avium for 6 h results in 90% inhibition of bacterial growth. The uninfected Mphi adhere to M. avium-infected apoptotic, but not to nonapoptotic M. avium-infected Mphi, suggesting a specific interaction between apoptotic and nonapoptotic Mphi. PCD of the host Mphi also prevents the release of intracellular components and the spread of the mycobacterial infection. Once the apoptotic infected Mphi reach the necrotic stage, mycobacteria and other intracellular material are released; the latter suffice to support extracellular mycobacterial replication. Necrosis of M. avium-infected Mphi is significantly augmented by the transglutaminase inhibitors dansyl-cadaverine and cystamine, indicating that apoptosis of Mphi is dependent on the extent of cross-linking of cell proteins by transglutaminases. Consequently, transglutaminase inhibitors accelerate the release of mycobacteria and intracellular components from the infected Mphi into the medium. These findings indicate that PCD of M. avium-infected Mphi is an important defense mechanism, preventing the spread of infection by sequestering the mycobacteria and by contributing to their demise by activation of newly recruited uninfected Mphi.

A new role for interleukin-7 in the induction of LFA-1 and VLA-4 adhesion molecules in Phorbol 12myristate 13acetate activated CD4+ CD23+ T-cell subset.

BACKGROUND: The low affinity receptor for IgE, CD23, has been described in several pathological conditions. However, the factors involved in the upregulation or downregulation of this receptor are still debated. METHODS AND RESULTS: We studied the effect of interleukin 7 (IL-7) on the expression of CD23 in normal PBT cells stimulated with PMA + Ca2. The data indicate that activated PB-T cultured in the presence of IL-7 showed an increased expression of CD23. The induction of IL-7 on CD23 production appears to be independent of IL-2, IL-4, IL-9, IL-15. Indeed, the addition of specific MoAbs anti-IL-2, IL-4, IL-9, IL-15 or anti-IL2R was unable to block the effect of IL-7 on CD23. The addition of IL-7 to a specific subset CD4+ CD23+ was able to augment the adhesiveness of T cells to parenchymal cell monalayers. The use of different cytokine (IL-2, IL-4, IL-9, IL-15) resulted in no increase of adhesiveness. In contrast the addition of IL-7 to a different T-cell subset (i.e. CD4+ CD23-) was unable to rescue the lack of adhesiveness observed in these cells. Blocking experiments with MHM6 MoAb were able to drastically reduce the adhesiveness observed in CD4+ CD23+ subsets. The presence of LFA-1 and VLA-4 adhesion molecules were responsible for the augmented adhesiveness of activated CD4+ CD23+ T cells cultured in the presence of IL-7. Blocking experiments with anti-LFA-1, VLA-4, anti-LFA-1beta plus VLA-4alpha MoAbs or anti-ICAM-1 MoAb added to the monolayers resulted in a complete inhibition of adhesion to parenchymal monolayers. In contrast, the addition of anti-IL-7 or anti-IL-7R MoAbs were able to block the augmented adhesiveness of CD4+ CD23+ cells to monolayers observed in the presence of IL-7. CONCLUSION: Taken together these findings point to the likelihood that IL-7 is responsible for the observed quantitative difference in the level of adhesion molecules and may open a new role of CD23 in the immune regulation.

Interleukin-7 modulates intracytoplasmatic CD23 production and induces adhesion molecule expression and adhesiveness in activated CD4+CD23+ T cell subsets.

The low-affinity receptor for IgE, CD23, has been described in several pathological conditions. However, the factors involved in the upregulation or downregulation of this receptor are still debated. We studied the effect of interleukin 7 (IL-7) on the production of CD23 in normal PBT cells stimulated with PMA + Ca2. The results demonstrate that cytoplasmic CD23 level was significantly augmented by costimulation with PMA + Ca2 plus IL-7 (1000 U/ml). Using an intracytoplamatic cytometric analysis, an accumulation of intracellular CD23 was observed at 48 hr in the presence of IL-7. This appears to have a profile different from the CD23 surface expression peaking at 72 hr of culture. We were also able to show that sCD23 was specifically increased by IL-7 and occurred with an early peak at 72 hr and a late peak at 120 hr of culture. The increased release and the biphasic production of sCD23 may reside in an accelerated degradation of the receptor due to an excessive accumulation of it. Restimulation of CD4+ T cells with PMA + Ca2 without IL-7 changed the profile of sCD23 production showing a second peak at 144 hr of culture. The induction of IL-7 on CD23 production appears to be independent of IL-2, IL-4, IL-9, and IL-15. Indeed, the addition of specific mAbs anti-IL-2, -IL-4, -IL-9, -IL-15, or anti-IL-2R was unable to block the effect of IL-7 on CD23. The addition of IL-7 to specific subset CD4+CD23+ was able to augment the adhesiveness of T cells to parenchymal cell monolayers. The use of different cytokine (IL-2, IL-4, IL-9, IL-15) resulted in no increase of adhesiveness. In contrast, the addition of IL-7 to a different T cell subset (i.e., CD4+CD23-) was unable to rescue the lack of adhesiveness observed in these cells. The adhesion molecules LFA-1 and VLA-4 were responsible for the augmented adhesiveness of activated CD4+CD23+ T cells cultured in the presence of IL-7. Blocking experiments with anti-LFA-1beta, VLA-4alpha, anti-LFA-1beta plus VLA-4alpha mAbs, or anti-ICAM-1 mAb added to the monolayers resulted in a complete inhibition of adhesion to parenchymal monolayers. In contrast, the addition of anti-IL-7 or anti-IL-7R mAbs was able to block the augmented adhesiveness of CD4+CD23+ cells to monolayers observed in the presence of IL-7. A significant augmentation of LFA-1 and VLA-4 was observed in cells cultured in the presence of IL-7. Taken together these findings point to the likelihood that IL-7 is responsible for the observed quantitative difference in the level of adhesion molecules and may open a new role of CD23 in the immune regulations.

Nuclear phosphoinositide-specific phospholipase C, phosphatidylinositol 4,5-bisphosphate and protein kinase C during rat spermatogenesis.

Presence and intracellular distribution of phosphoinositide-specific phospholipase C, phosphatidylinositol 4,5-bisphosphate and protein kinase C have been investigated in rat maturing germ cells and spermatozoa. The isoforms beta 1 and gamma 1 of phosphoinositide-specific phospholipase C were immunologically identified and found to be predominantly nuclear or cytoplasmic and nuclear, respectively. The two enzymes were present in the maturing cell lineage of the seminiferous tubule, except for the nucleus of late spermatids, and absent in spermatozoa, in which, however, a phosphoinositide-specific phospholipase C activity persisted, due to yet uncharacterized enzyme(s). Protein kinase C paralleled these developmental changes, and was completely down-regulated in both total cell homogenates and isolated nuclei obtained from spermatozoa. On the contrary, phosphatidylinositol 4,5-bisphosphate, present at the nuclear level in all cell types, accumulated in the nuclei of late spermatids and spermatozoa. These data support the contention that the spermatozoon nucleus stores a lipid-dependent signaling apparatus which could be reactivated either during sperm maturation or at fertilization.

CD23 expression in activated human T cells is enhanced by interleukin-7.

Despite evidence for the expression of the low-affinity Fc receptor for IgE (Fc epsilon RII/CD23) in several pathological conditions, the role played by CD23 in normal human T cells is still unclear. We studied the effect of a stomal-derived cytokine, interleukin (IL-7), on the expression of CD23 in human T cells stimulated with 10 micrograms/ml phytohemagglutinin (PHA). The results demonstrate that IL-7 did not induce CD23 expression in the resting T cells. However, PHA-induced CD23 expression was enhanced by costimulation with IL-7 (1,000 U/ml). Cytofluorometric analysis revealed that CD23 expression in activated T cells was enhanced by the addition of IL-7 (from 2% to 18%). It was also observed that the effect of IL-7 on CD23 expression is exclusively on CD4+ T cells. The enhanced expression of CD23 was blocked by an anti-IL-7 monoclonal antibody (mAb), but not by IL-2 and IL-4 mAbs. This suggests that IL-7 is a potent regulatory cytokine capable of acting independently of IL-2 and IL-4 in the expression of CD23. Northern blot analysis showed an increase in CD23 mRNA when activated T cells were cultured in the presence of IL-7. A significant increase in receptor numbers on activated T cells was detected by Scatchard analysis when IL-7 was added to the cell cultures. The induction of CD23 expression by IL-1, IL-3, IL-4, IL-5, IL-6, interferon-8 and OKT3 on PHA-activated T cells was not of the same magnitude as observed in the presence of IL-7. These results demonstrate a selective induction of CD23 expression on activated human T cells cultured in the presence of IL-7. These data indicate that the stromal-derived growth factor points to an important role of CD23 in the regulatory network of the immune response.

Changes of nuclear protein kinase C activity and isotype composition in PC12 cell proliferation and differentiation.

To establish whether protein kinase C was involved in the nuclear events underlying cell differentiation and proliferation, rat pheochromocytoma PC12 cells, serum-starved for 24 h, were treated with either differentiating doses of nerve growth factor or high serum concentrations, which represented a powerful mitogenic stimulus. Western blot analysis with isoform-specific antibodies, performed on whole cell homogenates, cytoplasms, and purified nuclei, showed that PKC isotypes alpha, beta I, beta II, delta, epsilon, eta, and zeta were expressed in PC12 cells and that all of them, except for beta I, were found at the nuclear level, variably modulated depending on the cell treatment. Compared to serum-stimulated cells, in which an early (1 day) and marked rise of protein kinase C activity was followed by a plateau, nerve growth factor-treated cells showed a progressive increase of protein kinase C activity coincident with the onset and maintenance of the differentiated phenotype. Western blot analysis of nuclei isolated from fully differentiated cells demonstrated an increase of protein kinase C alpha, paralleled by enhanced phosphotransferase activity along with the nerve growth factor treatment, and complete loss of the delta isotype. In contrast, in nuclei of proliferating PC12 cells, after an early but modest increase at 1 day of mitogenic stimulation, protein kinase C activity reached a plateau. Isotype-specific analysis indicated a concomitant increase of protein kinase C beta II, delta, and zeta and the appearance of protein kinase C epsilon and eta at the nuclear level. Considering the relative intensity of the cytoplasmic and nuclear immunoreactive bands under the three conditions examined, clear-cut translocation to the nucleus occurred for PKC epsilon and eta in serum-stimulated cells. Additional nuclear accumulation of PKC by translocation from the cytoplasm was prominently induced for the zeta isoform after mitogenic stimulation and for PKC alpha during prolonged NGF treatment. Our data suggest that nuclear translocation and selective activation of distinct protein kinase C isoforms play a relevant role in the control of proliferation and differentiation of the same cell type and that nuclear protein kinase C is crucial to the induction and persistence of the differentiated neuronal phenotype of PC12 cells.

Validity of ultrasonography for postoperative monitoring in pediatric urology.

PURPOSE: We studied the validity of ultrasonography for short-term followup in pediatric urology. MATERIALS AND METHODS: The study group comprised 137 children (187 urinary tracts) undergoing surgery at our hospital for congenital urological pathology between February 1982 and July 1992. The study protocol designed to evaluate urinary tract dilation postoperatively and monitor its progress, included ultrasound at discharge from the hospital, and repeat ultrasound between days 20 and 30, and days 45 and 60. Diuretic renography or excretory urography was indicated when urinary tract dilatation showed no signs of regressing or had increased on 2 consecutive evaluations. Ultrasound of the urinary tract was done to evaluate variations in the grade of dilatation of the pelves, calices, infundibula and ureters, and grade of hydronephrosis. RESULTS: Variations in the grade of dilatation of the infundibula and ureters were early sensitive indicators of the absence of obstruction. Using this protocol only 15 of the 187 urinary tracts (8%) corrected surgically needed further evaluation for suspected iatrogenic stenosis, including 3 with obstruction that required reoperation. No other cases of obstruction were detected during long-term followup. CONCLUSIONS: A series of sonographic evaluations performed within a short period and the greater significance attributed to more specific parameters, such as grade of dilatation of the infundibula and ureters, make ultrasound a valid means of monitoring urological cases postoperatively.

Identification of PI-PLC beta 1, gamma 1, and delta 1 in rat liver: subcellular distribution and relationship to inositol lipid nuclear signalling.

The subcellular distribution of PI-PLC beta 1, gamma 1, and delta 1 has been investigated in rat liver by western blot and immunohistochemical analysis with a panel of isoform-specific antibodies. The data obtained in situ on cryo-sectioned tissue indicate that PI-PLC beta 1 is predominantly nuclear, while gamma 1 is largely cytoplasmic and delta 1 is sharply restricted to the cytoplasm. In fractionation experiments, the Western blot analysis indicated that the recovery of the nuclear isoforms beta 1 and gamma 1 was not affected by the removal of the nuclear membrane, and that the two enzymes persisted in nuclear matrix and lamina, obtained after nuclease digestion and extraction with high salt and detergent. The assay of the phosphodiesterase activity in different cell fractions correlates with the observed relative abundance of the enzymes, and specific inhibition with neutralizing anti-beta 1 and -gamma 1 isoforms confirms that these are the enzymes active at the nuclear level. These results demonstrate that in rat liver cells, as in other cell types, different members of the PI-PLC family show a discrete intracellular distribution, and suggest that PI-PLC beta 1 and gamma 1 play a central role in modulating the nuclear phosphoinositide cycle.

Dysregulation of interleukin-7 receptor may generate loss of cytotoxic T cell response in human immunodeficiency virus type 1 infection.

Virus-specific cytotoxic T lymphocytes (CTL) play a crucial role in modulating an immune response against human immunodeficiency type 1 (HIV-1) infection. The generation of effector cytotoxic cells from CTL precursors involves intricate interactions with antigen via T cell receptors (TcR) and soluble cytokines. Interleukin (IL)-7 can affect T cell maturation and differentiation. Here we report on a group of five HIV-1-positive individuals who tested negative for env- and gag-specific CTL activity. When exogenous recombinant human IL-7 was added as a stimulus to the cultures, none (0/5) of the CTL-negative individuals exhibited a CTL response. Individuals that were negative for HIV-1-specific CTL activity were found to lack IL-7 receptor (IL-7R) on CD8+ cells with a comparable reduction on CD4+ cells. Increased shedding of IL-7R in the culture supernatant was observed. A significant reduction in receptor number was detected by binding of 125I-labeled IL-7 and Scatchard analysis. The lack of IL-7R is probably not due to endogenous IL-7, since it was not detectable in the culture supernatants of the patients studied. HIV-1 proteins may cause down-modulation of IL-7R expression, either by producing an insufficient number of molecules or by rapid decay of IL-7R on T cells. These changes may alter the cells' capability to respond to the IL-7 growth signal, resulting in CTL failure and subsequent mishandling of the virus.

Interleukin 2-independent interleukin 7 activity enhances cytotoxic immune response of HIV-1-infected individuals.

Virus-specific cytotoxic T lymphocytes (CTLs), which kill virus-infected cells, are thought to be a major host defense against viral infections. The addition of interleukin 7 (IL-7) at the onset of mitogen-stimulated cultures resulted in a marked (up to threefold) augmentation of env-specific cytotoxicity in human immunodeficiency virus type 1 (HIV-1)-infected individuals (p < 0.001). Addition of IL-7 on day 3 or 5 produced a significant but lesser augmentation of CTL response as compared to day 0. The IL-7-induced proliferative response and augmentation of cytotoxic activity was time and dose dependent, with an optimal IL-7 concentration of 1000 U/ml. Cell surface phenotypic analysis of CTL effector cells indicates that IL-7 primarily affects the proliferation of CD8+ T cells. Anti-IL-2 monoclonal antibody (MAb) substantially inhibited the proliferative effect of IL-2, but did not affect the proliferative effect of IL-7. Endogenous IL-2-induced generation of cytotoxic T cells was blocked by MAbs to IL-2 or IL-2R. The addition of IL-7 restored the process of conversion of precursor CTLs (pCTLs) to mature CTLs (mCTLs) and significantly enhanced specific cytolytic activity. It appears that IL-7 is a potent regulatory cytokine capable of acting independently of IL-2 in mitogen-specific activation of pCTLs to mCTLs. These data suggest that IL-7 should be considered as a potential therapeutic approach in AIDS and other infectious diseases in which CTL response declines.