Intracellular immunization: antibody targeting to subcellular compartments.
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Journal Article↗
Biomedical subjects
A Cattaneo
Publications and source records attributed to A Cattaneo.
Redox state of single chain Fv fragments targeted to the endoplasmic reticulum, cytosol and mitochondria.
In this paper we have engineered the targeting of ScFv fragments to mitochondria and demonstrated that this can occur efficiently. This extends the range of subcellular compartments where antibody domains can be targeted in order to interfere with the action of the corresponding antigen. Moreover, we have compared the redox state of ScFv fragments targeted to the secretory compartment, the cytosol and the mitochondria, and demonstrated that cysteine residues in ScFv targeted to the secretory compartments and to the mitochondria are oxidized. On the contrary, cytosolic antibody domains are expressed in a reduced state, which is probably the reason for their lower expression levels. These pitfalls, however, do not prevent their successful utilization for intracellular immunization.
Amino Acid Sequence↗
Fetal heart screening in low-risk pregnancies.
The aim of this study was to assess whether a screening program for fetal cardiac malformations is justified in a low-risk population, and which factors influence its accuracy. The fetal heart was evaluated in 7024 pregnant women at 20-22 weeks, and evaluation was repeated at a more advanced gestational age in 9% of cases. Cardiological follow-up was continued postnatally until 2 years of age. The overall prevalence of cardiac anomaly was 0.93%. The incidences of major and minor defects were 0.44% and 0.48%, respectively. There were 23 true positives (0.33%): in 20 cases, the diagnosis was made in the second trimester, and 13 women (65%) chose termination of pregnancy. Seventeen of the 20 cases identified in the second trimester were serious malformations. There were 42 false negatives (0.60%). Of these, 12 had signs of cardiac dysfunction at birth or within the 1st month of life, and three of them died as a result of their cardiac anomaly. There were eight false positives (0.11%), all of a minor type. Six abnormal karyotypes, out of a total of 21 performed in the true-positive group (28.5%), were found. In addition, five of the 42 newborns in the false-negative group had trisomy 21. The overall sensitivity was 35.4%, and 61.3% for major defects. The accuracy in two distinct periods was estimated because the level of experience of the operators was different: sensitivity was 45.2% in period 1 (1986-88) (77.8% for major defects) and 26.5% in period 2 (1989-92) (52.9% for major defects). We conclude that a fetal heart screening program in the obstetric population is justified. It defines a high-risk group for karyotyping, allows planning of delivery in a tertiary center or the choice of terminating the pregnancy for the parents and appears to have a positive cost-benefit ratio. A crucial factor is the level of training and experience of the operators, who need specific teaching support.
Adolescent↗
Identifying a putative common binding site shared by substance P receptor and an anti-substance P monoclonal antibody.
Substance P G-protein coupled receptor and the antigen recognition site of a monoclonal antibody raised against substance P share a stretch of five contiguous identical amino acids. This observation prompted us to build an atomic model of both the receptor and the antibody and to analyse their common features. In particular, we report here that a pocket of similar size and composition is present in both proteins, strongly suggesting a similarity in the mode of binding of both macromolecules to substance P. From the analysis of our models, the available data on the mode of binding of the antibody to substance P and recent data on substance P receptor mutants, we concluded that the pocket is very likely to be involved in binding of the C-terminal 'message sequence' of the tachykinin. This allowed us to suggest specific site-directed mutants of the receptor which should shed some light on the mechanism of peptide recognition by G-protein coupled receptors.
Amino Acid Sequence↗
Squamous cell carcinoma arising in vulval lichen sclerosus: a longitudinal cohort study.
Histological changes of lichen sclerosus (LS)--a chronic inflammatory disease--are frequently found in association with squamous cell carcinoma (SCC) of the vulva, suggesting that women with this disorder are at increased risk. However, follow-up studies have been less convincing, showing that the vast majority of these patients do not go on to develop cancer. In this study, a series of 211 women affected by histologically demonstrated vulval LS were treated with topical therapy (testosterone, clobetasol) and followed prospectively by repetitive vulval examination. Three patients developed SCC of the vulva (two invasive, one in situ) at the sites affected by LS during an average follow-up period of 1 year and 8 months. Compared with the reference population, the number of cases of invasive SCC detected significantly exceeded the number estimated to occur in a comparable age-matched group. The standardized incidence rate of vulval SCC in the LS cohort was 317 (95% CI 35.7-1146.2). Cumulative risk was 14.8% (0.06% in the general female population), with a relative risk of 246.6. In conclusion, these data support the view that LS is a precursor of SCC, although characterized by slight tendency to evolve to carcinoma. Medical treatment of LS, although useful in the control of severity of disease, did not seem to be able to prevent the evolution to malignancy.
Administration, Topical↗
[Histopathologic problems in non neoplastic disease of the vulva].
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Female↗
The use of phage display in neurobiology.
Phage display is a new technique that is used extensively in molecular biology to study protein-protein interaction, receptor- and antibody-binding sites, to produce monoclonal antibodies against diverse antigens, some of which are too well conserved for the production of monoclonal antibodies by traditional means, and to improve or modify the affinity of proteins for their binding partners. This technique could have many applications in neurobiology. This review describes the background to the technique, and illustrates a number of possible uses in neurobiology, ranging from the production of antibodies to non-immunogenic proteins and to those that are available as cloned DNA sequences only, to the detailed study of receptor-ligand interaction using either ligands, their receptors or neutralizing antibodies.
Antibodies, Monoclonal↗
Idiopathic vulvodynia. Clinical evaluation of the pain threshold with acetic acid solutions.
Idiopathic vulvodynia is vulvar discomfort in which a diagnosis has not yet been established. As in other idiopathic pain syndromes, the involvement of primary afferent fibers (PAFs) has been postulated as playing a role in the pathogenesis and maintenance of idiopathic vulvodynia. Capsaicin induces the release of substance P (SP) by PAFs, producing vasodilation and increasing vascular permeability (neurogenic inflammation). Likewise, it has been shown that acid solutions can stimulate PAFs with the release of SP. To evaluate the pain threshold in women with idiopathic vulvodynia, 10 patients with vulvar pain but without significant vulvar physical changes and 10 asymptomatic controls received topically applied acetic acid solutions with increasing hydrogenionic concentrations (pH 3, 2.5, 2, 1.5, 1.2). Results related to pain threshold and pain time monitoring and intensity were analyzed with Fisher's exact and Wilcoxon's tests, respectively. Our data suggest that in idiopathic vulvodynia the pain threshold for acid solutions is decreased, probably in relation to increased sensitivity of PAFs involved in the transduction of painful signals.
Acetates↗
Production of type-1 and type-2 cytokines by peripheral blood mononuclear cells of psoriatic patients.
We investigated the function of peripheral blood mononuclear cells (PBMC) in 16 patients with active psoriasis, in 15 patients with static psoriasis and in 27 healthy volunteers, by examining in vitro proliferation and antigen- and mitogen-stimulated production of interleukin-2 (IL-2) and IL-4. Plasma levels of the neuropeptide substance P were also determined. Defective alloantigen (ALLO)- and phytohaemagglutinin (PHA)-stimulated IL-2 production was detected in 42% and in 45% of psoriatic patients, respectively. The number of defective IL-2 responders was higher in static (60%) than in active (25%) psoriasis. The reduction of IL-2 responses in the former group was associated with an increase of IL-4 production. Thus PBMC of 66% of patients with static psoriasis but none of the patients with active psoriasis produced elevated amounts of PHA-stimulated IL-4. Variations of plasma substance P levels followed the same pattern of IL-4, being higher in static than in active psoriasis. These observations suggest a co-ordinated action of IL-4 and substance P as modulators of the clinical course of psoriasis. Our data show a possible correlation between the clinical evolution of psoriasis and the production of type-1 and type-2 cytokines, suggesting that the former may have a prominent role in the activation of psoriasis, while the latter may play a protective role.
Acute Disease↗
Massive vulvar edema in ovarian hyperstimulation syndrome. A case report.
BACKGROUND: Bilateral vulvar edema associated with severe ovarian hyperstimulation syndrome is described for the first time. CASE: A 28-year-old woman underwent gonadotropin-releasing hormone analogue and gonadotropin treatment for in vitro fertilization and embryo transfer. On day 18 of the cycle, the patient reported mild abdominal discomfort that became severe during the following four days. One the 22nd day of the cycle, bilateral ovarian enlargement and ascites were present. The vulva showed massive edema and fissures. Ovarian hyperstimulation syndrome (OHSS) therapy consisted of human albumin, lactated Ringer's solution and heparin. The vulvar edema was treated with topical hydrocortisone ointment, ice packs and topical gentamycin twice a day. After one week of treatment, the vulva was normal. CONCLUSION: We think that the vulvar edema in this case was the result of decreased oncotic pressure and increased hydrostatic pressure, as occur during OHSS.
Administration, Topical↗
Kangaroo mother method for low-birthweight infants.
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Body Temperature Regulation↗
Antibodies to nerve growth factor (NGF) prolong the sensitive period for monocular deprivation in the rat.
Neural plasticity in the visual cortex, as tested by changes in its functional organization induced by monocular deprivation (MD), is present only during a restricted period of postnatal development (critical period). To investigate whether this process of synapse strengthening depends upon NGF, we antagonized endogenous NGF during the critical period by implanting anti-NGF producing cells. Anti-NGF treated and control rats were monocularly deprived after the end of the critical period. In anti-NGF treated but not in control rats MD was still effective. We conclude that antagonism of endogenous NGF prolongs the critical period, possibly by delaying the process of synapse consolidation in the visual cortex.
Animals↗
The use of the RACE method to clone hybridoma cDNA when V region primers fail.
The technique of V region PCR to clone antibody V regions from hybridomas has been extensively used. However, in addition to, or even instead of, cloning the V regions with the desired specificity, myeloma cell derived V regions, V regions which are the result of non-productive rearrangements, and also V regions which are productive but which do not recognise the antigen of interest, may be isolated. In this paper we describe a comparison of the use of V region PCR and a modification of the RACE technique to clone the V region of the anti-NGF hybridoma, alpha D11. This hybridoma has heavy and light chain V regions which are refractory to amplification with V region primers, but which are easily amplified using RACE, a PCR based procedure which is independent of the variability within the V regions.
Animals↗
Monoclonal antibodies to nerve growth factor affect the postnatal development of the visual system.
Exogenous supply of nerve growth factor (NGF) prevents the effects of monocular deprivation. This suggests that visual afferents may be competing for an endogenous neurotrophic factor, related to NGF, whose production by postsynaptic cells depends on the activity of afferent fibers. To test the hypothesis that endogenous NGF may play a role in the functional and anatomical development of the rat geniculo cortical system, the physiological action of NGF in the rat visual system was antagonized by using two independent monoclonal antibodies which neutralize NGF (alpha D11 and 4C8). To provide a continuous supply of antibodies during the period of visual cortical plasticity, alpha D11 or 4C8 antibody-producing hybridoma cells were implanted in the lateral ventricle of rats at postnatal day 15. This resulted in dramatic alterations of two of the most important parameters characterizing the functional development of the visual system, namely, visual acuity and binocularity of cortical neurons and in shrinkage of cells in the lateral geniculate nucleus. This demonstrates that the action of endogenous NGF is necessary for the normal functional and anatomical development of the geniculocortical system.
Animals↗
Low-affinity nerve growth factor receptor is expressed during testicular morphogenesis and in germ cells at specific stages of spermatogenesis.
Nerve growth factor (NGF) is essential for neuronal development and differentiation. Recent reports have shown that its low-affinity receptor (LNGFR) is expressed and developmentally regulated in a broad range of embryonic and adult tissues outside the nervous system, although the functions of the receptor in such tissues remain unknown. Recently, NGF and LNGFR have been detected in adult mouse, rat, and human testis. The results of the present work demonstrate that LNGFR is expressed much before the onset of spermatogenesis in both mouse and rat testis. In situ hybridization shows that the mRNA for LNGFR is expressed in the peritubular cells of the embryonic mouse testis. Immunohistochemical analysis of the rat testis shows LNGFR-expressing cells to be scattered in the intertubular compartment in the embryonic testis, and to become organized in a cellular layer that surrounds myoid cells of the seminiferous tubules during postnatal development. Furthermore, in peripuberal and adult mouse and rat testis we have identified the expression of an abundant and shorter mRNA of 3.2 kb that cross hybridizes to the low-affinity NGF receptor transcript (3.7 kb). This shorter mRNA species, which appears at the beginning of spermatogenesis in the adult, has been identified by in situ hybridization and by Northern blot with RNA isolated from homogeneous populations of meiotic germ cells to be expressed by pachytene spermatocytes and round spermatids. Our results suggest a complex developmental role for LNGFR during testicular morphogenesis and identify the expression, at specific stages of spermatogenesis, of a new germ cell-specific transcript homologous to the receptor RNA.
Animals↗
Intracellular immunization with cytosolic recombinant antibodies.
We report the application of a strategy to inactivate cellular proteins in vertebrate cells based on the intracellular expression of immunoglobulin genes. We have selected, in this instance, the p21 protein, encoded by the ras proto-oncogene, as a target protein. The variable regions of the neutralizing anti-p21ras monoclonal antibody Y13-259 were cloned in vectors for the expression of either the whole antibody molecule or its single-chain Fv fragment (ScFv) derivative. In order to target the recombinant antibodies to the cytosol, their hydrophobic leader sequence for secretion was mutated or deleted. When these proteins are expressed in the cytosol of Xenopus laevis oocytes they colocalize with the endogenous p21ras protein in the cytoplasmic face of the oocyte plasma membrane, and they markedly inhibit the H1 kinase activity induced by insulin. Moreover, cytosolic anti-p21ras ScFv fragments block the ensuing meiotic maturation. Thus the intracellular expression of both whole antibodies and antibody domains can be used to block a biological function.
Animals↗
Plasma neuropeptide levels in psoriasis.
The immune system is important in the pathogenesis of psoriasis and emotional stress has precipitated psoriasis in many patients. Neuropeptides, alpha-Melanocyte stimulating hormone (alpha-MSH), beta-endorphin, met-enkephalin and substance P (SP) act as immunomodulators, and their secretion increases during periods of stress. To see whether these neuropeptides themselves might be related to psoriasis and/or to the aggressiveness of the disease, we evaluated the plasma neuropeptide levels in 13 patients with active psoriasis (patients with new lesions and/or pre-existing lesions that had become larger during the month before the study), in 11 patients with stable psoriasis and in 10 healthy controls. Plasma concentrations of neuropeptides were evaluated by RIA (immunoradiometric assay for beta-endorphin). Data were compared by the Student t-test for unpaired data. There were no significant differences between the plasma levels of any of the neuropeptides between active psoriatic patients and stable psoriatic patients, nor between the plasma levels of neuropeptides of psoriatic patients and those of control subjects. It seems unlikely that circulating neuropeptide levels are of primary importance in the manifestation of the psoriatic skin lesions.
Adult↗
Topical calcipotriol for psoriasis--an immunohistologic study.
The aim of the present study was to investigate the distribution of Langerhans cells and T cells in the lesions and also the phenotypic expression of markers of activation on lesional T cells and keratinocytes, before and after 2 weeks of topical treatment of 7 psoriatic patients with calcipotriol. Before treatment, the infiltrate was composed mainly of T cells and there was decreased expression of CD1 on the intra-epidermal Langerhans cells. ICAM-1 and EGF receptor were present throughout the epidermis, but keratinocytes expressing Transferrin receptor were detected only in the basal layer. After 14 days of calcipotriol therapy, there were significantly fewer CD4T cells in the dermis and an increased number of intraepidermal CD1 + Langerhans cells. ICAM-1 expression on lesional keratinocytes was reduced in all patients, but the expression of EGF receptor was decreased in 3 patients only, and Transferrin receptor expression on keratinocytes had not changed. All these changes were concurrent with moderate clinical improvement of the lesions. The results suggest that in the early stages of the clinical response to calcipotriol there is an immunomodulating effect of the drug associated with variable decreases in keratinocyte expression of markers of activation.
Administration, Topical↗