Safety and efficacy of immunotherapy with the recombinant B-cell epitope-based grass pollen vaccine BM32.

BACKGROUND: BM32 is a grass pollen allergy vaccine based on recombinant fusion proteins consisting of nonallergenic peptides from the IgE-binding sites of the 4 major grass pollen allergens and the hepatitis B preS protein. OBJECTIVE: We sought to study the safety and clinical efficacy of immunotherapy (allergen immunotherapy) with BM32 in patients with grass pollen-induced rhinitis and controlled asthma. METHODS: A double-blind, placebo-controlled, multicenter allergen immunotherapy field study was conducted for 2 grass pollen seasons. After a baseline season, subjects (n = 181) were randomized and received 3 preseasonal injections of either placebo (n = 58) or a low dose (80 µg, n = 60) or high dose (160 µg, n = 63) of BM32 in year 1, respectively, followed by a booster injection in autumn. In the second year, all actively treated subjects received 3 preseasonal injections of the BM32 low dose, and placebo-treated subjects continued with placebo. Clinical efficacy was assessed by using combined symptom medication scores, visual analog scales, Rhinoconjunctivitis Quality of Life Questionnaires, and asthma symptom scores. Adverse events were graded according to the European Academy of Allergy and Clinical Immunology. Allergen-specific antibodies were determined by using ELISA, ImmunoCAP, and ImmunoCAP ISAC. RESULTS: Although statistical significance regarding the primary end point was not reached, BM32-treated subjects, when compared with placebo-treated subjects, showed an improvement regarding symptom medication, visual analog scale, Rhinoconjunctivitis Quality of Life Questionnaire, and asthma symptom scores in both treatment years. This was accompanied by an induction of allergen-specific IgG without induction of allergen-specific IgE and a reduction in the seasonally induced increase in allergen-specific IgE levels in year 2. In the first year, more grade 2 reactions were observed in the active (n = 6) versus placebo (n = 1) groups, whereas there was almost no difference in the second year. CONCLUSIONS: Injections of BM32 induced allergen-specific IgG, improved clinical symptoms of seasonal grass pollen allergy, and were well tolerated.

The immune profile induced is crucial to determine the effects of immunocastration over gonadal function, fertility, and GnRH-I expression.

PROBLEM: Immunocastration or vaccination against the GnRH-I hormone is a promising alternative to reproductive control in different animal species. Given the low immunogenicity of this hormone, the use of adjuvants becomes necessary. METHOD OF STUDY: This study evaluated the effects of three adjuvants that induce different immune response profiles over gonadal function, fertility, and expression of GnRH-I. Female mice (n = 6) were vaccinated at days 1 and 30 with a recombinant antigen for immunocastration and different adjuvants that induced preferentially Th1/Th2, Th2, and Th1 immune profiles. RESULTS: Th1/Th2 response is the most efficient to block reproductive activity in vaccinated animals, reducing the number of luteal bodies and pre-ovulatory follicles. Th2 and Th1/Th2 responses induced an increase in GnRH-I at the hypothalamus. CONCLUSION: The immune profile induced by different adjuvants is essential on the effects over fertility, gonadal function, and hypothalamic GnRH-I expression in immunocastrated animals.

Adjuvant treatment with crude rhubarb for patients with systemic inflammation reaction syndrome/sepsis: a meta-analysis of randomized controlled trials.

OBJECTIVE: The objective of this study is to evaluate the benefits of adjuvant treatment with crude rhubarb in patients with systemic inflammation reaction syndrome/sepsis by conducting a meta-analysis. METHODS: We conducted a systematic literature search of medical electronic databases (up to October 2013). Only randomized controlled trials (RCTs) assessing adjuvant treatment with crude rhubarb in septic patients were included. RESULTS: A total of 15 RCTs with 869 patients were identified. Pooled analysis showed that interleukin 6 (standardized mean differences [SMDs], -1.30; 95% confidence intervals [CIs], -1.94 to -0.66), tumor necrosis factor α (SMD, -0.95; 95% CI, -1.55 to -0.36), procalcitonin (SMD, -1.50; 95% CI, -2.20 to -0.80), von Willebrand factor (mean differences [MDs], -144.11; 95% CI, -253.87 to -34.35), prothrombin time (MD, -2.38; 95% CI, -2.67 to -2.10), acute physiology and chronic health evaluation II scores (MD, -4.51; 95% CI, -5.30 to -3.73), and gastrointestinal dysfunction (risk ratio, 0.28; 95% CI, 0.16-0.49) were significantly reduced after treatment with crude rhubarb. Platelet number (MD, 58.16; 95% CI, 51.16-65.15) was significantly increased. However, crude rhubarb therapy did not significantly reduce 28-day mortality (risk ratio, 0.60; 95% CI, 0.36-1.00) compared with the usual treatment. CONCLUSIONS: Adjuvant treatment with crude rhubarb appears to have additional benefits in septic patients. Antiinflammation and anticoagulant/antiaggregant properties may be its potential mechanism.

IGRP and insulin vaccination induce CD8+ T cell-mediated autoimmune diabetes in the RIP-CD80GP mouse.

Autoimmune diabetes is characterized by autoantigen-specific T cell-mediated destruction of pancreatic islet beta cells, and CD8(+) T cells are key players during this process. We assessed whether the bitransgenic RIP-CD80 x RIP-LCMV-GP (RIP-CD80GP) mice may be a versatile antigen-specific model of inducible CD8(+) T cell-mediated autoimmune diabetes. Antigen-encoding DNA, peptide-loaded dendritic cells and antigen plus incomplete Freund's adjuvant were used for vaccination. Of 14 pancreatic proteins tested by DNA vaccination, murine pre-proinsulin 2 (100% of mice; median time after vaccination, 60 days) and islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) (77%, 58 days) could induce diabetes. Vaccination with DNA encoding for zinc transporter 8, Ia-2, Ia-2ß, glutamic acid decarboxylase 67 (Gad67), chromogranin A, insulinoma amyloid polypeptide and homeobox protein Nkx-2.2 induced diabetes development in 25-33% of mice. Vaccination with DNA encoding for Gad65, secretogranin 5, pancreas/duodenum homeobox protein 1 (Pdx1), carboxyl ester lipase, glucagon and control hepatitis B surface antigen (HBsAg) induced diabetes in <20% of mice. Diabetes induction efficiency could be increased by DNA vaccination with a vector encoding a ubiquitin-antigen fusion construct. Diabetic mice had florid T cell islet infiltration. CD8(+) T cell targets of IGRP were identified with a peptide library-based enzyme-linked immunospot assay, and diabetes could also be induced by vaccination with major histocompatibility complex (MHC) class I-restricted IGRP peptides loaded on mature dendritic cells. Vaccination with antigen plus incomplete Freund's adjuvant, which can prevent diabetes in other models, led to rapid diabetes development in the RIP-CD80GP mouse. We conclude that RIP-CD80GP mice are a versatile model of antigen specific autoimmune diabetes and may complement existing mouse models of autoimmune diabetes for evaluating CD8(+) T cell-targeted prevention strategies.

Effect of preoperative fever-range whole-body hyperthermia on immunological markers in patients undergoing colorectal cancer surgery.

BACKGROUND: Previous studies have demonstrated beneficial immunological effects of fever-range whole-body hyperthermia (FR-WBH) as an adjunct to non-surgical cancer therapy. We conducted a study of preoperative FR-WBH in patients undergoing colorectal cancer surgery to evaluate perioperative, hyperthermia-induced immunomodulation. METHODS: The trial was conducted as a subject-blinded, controlled, randomized study. Subjects in the FR-WBH group (n=9) were treated with FR-WBH before operation under propofol sedation; the target core temperature was 39 (0.5)°C with 1 h warming and 2 h plateau phase. Subjects in the control group (n=9) were treated with propofol sedation only. Blood samples were acquired before and after treatment, after operation, and 24, 48 h, and 5 days after the end of surgery. The following parameters were measured: lipopolysaccharide (LPS)-induced tumour necrosis factor (TNF)-α, procalcitonin (PCT), interleukin (IL)-6/10, heat shock proteins (HSPs) 60, 70, and 90, human leucocyte antigen-DR (HLA-DR), and LPS-binding protein (LBP). RESULTS: HSPs were increased in the FR-WBH group after treatment [HSP60, 48 h postop: 143 (41)% vs 89 (42)%, P=0.04; HSP90, postop: 111 (33)% vs 64 (31)%, P=0.04; HSP70: P=0.40; FR-WBH vs control, P-values for area under the level/time curve]. TNF-α levels were elevated after surgery in the control group and remained near baseline in the FR-WBH group [24 h postop: 73 (68)% vs 151 (72)%, P=0.04]. PCT increased in both groups 24 h after surgery; in the control group, this increase was significantly higher (P=0.02). There were no significant differences for IL, HLA-DR, or LBP. CONCLUSIONS: The immune system to react to surgical stress, as measured by a panel of laboratory indicators, might be improved by preoperative FR-WBH.

Central administration of aminoprocalcitonin inhibits food intake and stimulates the hypothalamic-pituitary-adrenal axis in rats via the corticotrophin-releasing factor system.

Aminoprocalcitonin (N-PCT), a neuroendocrine peptide derived from procalcitonin, reduces food intake and body weight when administered centrally in rats. We have recently shown that N-PCT is expressed in brain areas known to be involved in energy homeostasis, including the paraventricular nucleus (PVN) of the hypothalamus, which contains a prominent population of corticotrophin-releasing factor (CRF)-synthesising neurones. CRF plays a pivotal role in the regulation of the hypothalamic-pituitary adrenal (HPA) axis and food intake. However, little is known about functional interactions of N-PCT and CRF. In the present study, we found endogenous N-PCT protein in the rat PVN. We also showed N-PCT immunoreactivity in PVN co-localised with NeuN, a neuronal marker, or glial fibrillary acidic protein, an astrocyte marker. Double staining immunohistochemistry revealed that N-PCT co-localised with CRF in parvocellular neurones of the PVN. Intracerebroventricular N-PCT administration increased CRF mRNA and content in the hypothalamus, suggesting that N-PCT stimulates the HPA axis and suppresses food intake and body weight via CRF-dependent pathways. In keeping with this, i.c.v. co-injection of D-Phe-CRF(12-41), a CRF receptor antagonist, significantly attenuated N-PCT-induced reduction in food intake and body weight in a dose-dependent manner. Furthermore, i.c.v. administration of N-PCT increased plasma adrenocorticotrophic hormone and corticosterone concentrations and induced the expression of Fos protein, a marker of neuronal activity, in parvocellular CRF neurones. These data collectively support the hypothesis that N-PCT inhibits food intake and body weight and stimulates the HPA axis via CRF-mediated pathways.

Immunoneutralization of the aminoprocalcitonin peptide of procalcitonin protects rats from lethal endotoxaemia: neuroendocrine and systemic studies.

Severe sepsis and septic shock are an important cause of mortality and morbidity. These illnesses can be triggered by the bacterial endotoxin LPS (lipopolysaccharide) and pro-inflammatory cytokines, particularly TNF-α (tumour necrosis factor-α) and IL (interleukin)-1ß. Severity and mortality of sepsis have also been associated with high concentrations of N-PCT (aminoprocalcitonin), a 57-amino-acid neuroendocrine peptide derived from ProCT (procalcitonin). Previous studies in a lethal model of porcine polymicrobial sepsis have revealed that immunoneutralization with IgG that is reactive to porcine N-PCT significantly improves short-term survival. To explore further the pathophysiological role of N-PCT in sepsis, we developed an antibody raised against a highly conserved amino acid sequence of human N-PCT [N-PCT-(44-57)]. This sequence differs by only one amino acid from rat N-PCT. First, we demonstrated the specificity of this antibody in a well-proven model of anorexia induced in rats by central administration of human N-PCT-(1-57). Next we explored further the therapeutic potential of anti-N-PCT-(44-57) in a rat model of lethal endotoxaemia and determined how this immunoneutralization affected LPS-induced lethality and cytokine production. We show that this specific antibody inhibited the LPS-induced early release of TNF-α and IL-1ß and increased survival, even if treatment began after the cytokine response had occurred. In addition, anti-N-PCT-(44-57) may increase long-term survival in LPS-treated rats by up-regulating the late production of counter-regulatory anti-inflammatory mediators such as ACTH (adrenocorticotropic hormone) and IL-10. In conclusion, these results support N-PCT as a pro-inflammatory factor in both the early and the late stages of lethal endotoxaemia, and suggest anti-N-PCT as a candidate for septic shock therapy.

Isolation, cloning, and characterization of the 2S albumin: a new allergen from hazelnut.

SCOPE: 2S albumins are the major allergens involved in severe food allergy to nuts, seeds, and legumes. We aimed to isolate, clone, and express 2S albumin from hazelnut and determine its allergenicity. METHODS: 2S albumin from hazelnut extract was purified using size exclusion chromatography and RP-HPLC. After N-terminal sequencing, degenerated and poly-d(T) primers were used to clone the 2S albumin sequence from hazelnut cDNA. After expression in Escherichia coli and affinity purification, IgE reactivity was evaluated by Immunoblot/ImmunoCAP (inhibition) analyses using sera of nut-allergic patients. RESULTS: N-terminal sequencing of a approximately 10 kDa peak from size exclusion chromatography/RP-HPLC gave two sequences highly homologous to pecan 2S albumin, an 11 amino acid (aa) N-terminal and a 10 aa internal peptide. The obtained clone (441 bp) encoded a 147 aa hazelnut 2S albumin consisting of a putative signal peptide (22 aa), a linker peptide (20 aa), and the mature protein sequence (105 aa). The latter was successfully expressed in E. coli. Both recombinant and natural 2S albumin demonstrated similar IgE reactivity in Immunoblot/ImmunoCAP (inhibition) analyses. CONCLUSION: We confirmed the postulated role of hazelnut 2S albumin as an allergen. The availability of recombinant molecules will allow establishing the importance of hazelnut 2S albumin for hazelnut allergy.

Identification and localization of procalcitonin-like immunoreactivity in the rat hypothalamus.

Procalcitonin (PCT) is a 116-amino acid polypeptide physiologically produced, as the precursor protein of calcitonin (CT), in the parafollicular cells of the thyroid gland, but physiological functions and other major sources of PCT remains unclear. The distribution of PCT-like immunoreactivity (PCT-LI) in the rat hypothalamus was examined by immunohistochemistry using a monoclonal antibody raised against the mid-region of human PCT (60-77-amino acid fragment). This antibody cross-reacts well with rat PCT and immature CT, but it cross-react poorly with free mature CT. Abundant expression of PCT-LI was found in zones at the interface between brain and cerebrospinal fluid (CSF) such as the ependymal layer and ventral glia limitans (VGL). Double labeling of PCT and glial fibrillary acidic protein (GFAP) identified this population of small cells as astrocytes, possibly tanycytes, a type of specialized glial cell that interacts in neuroendocrine functional dynamics. The fibers of these cells extend to circumventricular organs (CVOs) and to astrocytes located inside the parenchyma of key autonomic regulatory hypothalamic areas, with highest densities in the supraoptic nucleus (SO), arcuate nucleus (Arc), area postrema (AP), median eminence (ME), medial preoptic nucleus, tuber cinereum, and accessory neurosecretory nuclei. No strongly labeled cells were found in the paraventricular nucleus. The wide distribution of PCT-LI in the hypothalamus, in close correspondence with previous mapping of CT receptors in the rat brain, suggests that PCT may influence a multitude of biological activities associated with the hypothalamic-pituitary axis.

[Adaptation of different modifications of enzyme immunoassay and immune blot assay to assess the candidate vaccine HIVREPOL against HIV/AIDS].

A preclinical trial of the vaccine HIVREPOL provided a complex of methods for assessing the identity and specific activity of vaccines against HIVIAIDS. The identity of "HIVREPOL" has been assessed by indirect enzyme immunoassay (EIA): the vaccine specifically binds the antibodies of the sera from HIV-infected individuals. Immune blot assay was the most informative method for assessing the identity of the candidate vaccine. The sera from HIVREPOL-vaccinated mice recognized the proteins gp41, p24, p55 of cultured HIV1 on "New-Lay-Blot1" strips. The bands corresponding to p24 were revealed in the line blots "Blot-HIV-1/2+O" and "INNO-LIA-HIV-Confirmation". The specific activity of the HIVREPOL vaccine was confirmed from the reactivity of sera of the mice vaccinated with recombinant proteins of the immunosorbents available in EIA test systems for the detection of HIV antibodies. Competitive EIA established the antigen-binding activity of sera from HIVREPOL-vaccinated mice against the native reference HIV-1 antigen.

Calcitonin gene related peptide and N-procalcitonin modulate CD11b upregulation in lipopolysaccharide activated monocytes and neutrophils.

OBJECTIVE: Circulating levels of calcitonin gene related peptide (CGRP) and calcitonin precursors, including procalcitonin (PCT) and its free aminopeptide N-procalcitonin (N-PCT), have been found dramatically increased in septic patients. PCT is known to attenuate the chemotaxis of monocytes in response to chemoattractants. This study examined whether CGRP and N-PCT modulate the LPS-induced expression of CD11b, which is one of the major integrins involved in monocyte and neutrophil chemotaxis during a response to microbial infections. DESIGN AND SETTING: In vitro cell culture study in the immunology laboratory of a university hospital. PARTICIPANTS: Healthy volunteers. MEASUREMENTS AND RESULTS: We assessed the effects of N-PCT and CGRP on CD11b expression on monocytes and neutrophils after LPS (2 ng/ml) or fMLP (10(-8) M) challenges. We used a human whole blood model, and measurements were made by flow cytometry. Both peptides in a dose-dependent manner decreased the LPS- and fMLP-induced rise in CD11b in monocytes and neutrophils. As these peptides are thought to act by raising cAMP, we also mimicked their effects with the use of rolipram and forskolin and found similar results. CONCLUSIONS: These findings are in line with recent studies demonstrating anti-inflammatory properties for this family of peptides. CGRP and calcitonin precursors may function as factors suppressing the propagation of inflammation through the inhibition of several processes involved during a response to a bacterial stimulus.

A cross-sectional study of workers with occupational exposure to petroleum derivatives.

Expression of human involucrin (hINV) a protein of the cornified cell envelope, was studied in the skin of gasoline storage workers, in order to evaluate the effects of the exposure to petroleum derivatives. A total of 25 forearm skin punch biopsies were carried out. Twenty of which were performed on exposed subjects and five on controls. The specimens were processed for immunohistochemistry and hINV expression was evaluated using an anti-hINV monoclonal antibody and the ABC technique. Percentage of immunolabeled keratinocytes was significantly higher in subjects exposed to gasoline with respect to the control sample. A premature hINV expression was detected both in suprapapillary and interpapillary keratinocytes. Such overexpression of hINV seems to be related to an attempt of increasing skin defence mechanism. Therefore it was concluded that also in absence of clinical skin manifestation the exposure to gasoline determines an involvement of keratinocytes on molecular basis.

[The antidiphtheria activity of the pre-EGF fragment]. / Antidifteriinaia aktivnost' fragmenta pre-EGF.

The DNA fragment, coding a part of the protein molecule--the precursor of the epidermal growth factor (pre-EGF76-208)--and containing the sequence of 133 N-end amino acid residues, was obtained with the use of gene engineering and molecular biological techniques. For this purpose a fraction of poly(A+) = RNA was isolated from the kidney of a newborn infant; on this fraction the "library" of cDNA fragments whose coding capacity corresponded to the required sequence of mRNA in the pre-EGF gene was obtained in the reaction of reverse transcription, conjugated with the polymerase chain reaction (PCR). After the determination of the nucleotide sequences in 11 fragments only 1 fragment which contained practically no mutations appearing in PCR as the result of amplifications was chosen. This fragment was used for the construction of a hybrid plasmid controlling the synthesis of hybrid fusion protein with the sequence of pre-EGF76-208. Fusion protein was synthesized with very low effectiveness, but the use of metallo-affinity chromatography permitted to isolate and purify it, its purity reaching 98%. Then its antitoxic activity was determined in the skin test on guinea pigs and found to be not less then 10(6) I.U./mg of protein.

Cloning of human keratolinin cDNA: keratolinin is identical with a cysteine proteinase inhibitor, cystatin A, and is regulated by Ca2+, TPA, and cAMP.

Keratolinin has been described as one of the precursor proteins of cornified cell envelope of keratinocytes. Using rabbit polyclonal anti-human keratolinin antibody, we isolated a cDNA clone of human keratolinin gene from a human Agt11 cDNA expression library that was constructed by random priming from poly(A)+RNA extracted from cultured normal human keratinocytes. Screening by rabbit anti-human keratolinin antibody detected one positive clone (HKL-1). The recombinant 12.5-kDa protein constructed from the clone reacted specifically with the anti-human keratolinin antibody. DNA sequence analysis revealed that HKL-1 clone was 448 bp long, and its putative amino acid sequence was identical with that of a human cysteine proteinase inhibitor, cystatin A. Western blot analysis showed that the commercially available recombinant cystatin A also reacted specifically with the anti-human keratolinin antibody. Northern blot analysis indicated that HKL-1 clone hybridizes with mRNA of about 0.5 kb, consistent with the size of the HKL-1 clone. The keratolinin mRNA was highly expressed in cultured human keratinocytes in high Ca2+ (1 mM); in low Ca2+ (0.05 mM), the keratolinin mRNA expression was significantly lower. Using SV40-transformed human keratinocytes (SVHK cells), we further analyzed the regulation of keratolinin mRNA. In low Ca2+ (0.05 mM), keratolinin mRNA in SVHK cells was marginally detectable. Upon shift to 1 mM calcium, keratolinin mRNA was markedly increased. The upregulation of keratolinin mRNA was also observed by the treatment of SVHK cells with 10 ng TPA per ml or 100 microM forskolin under low calcium conditions (0.05 mM). Our results indicate that keratolinin is identical with cystatin A, a cysteine proteinase inhibitor, and its expression is positively regulated by Ca2+, TPA, and forskolin.

Biosynthesis of prothrombin: intracellular localization of the vitamin K-dependent carboxylase and the sites of gamma-carboxylation.

Prothrombin is a vitamin K-dependent blood coagulation protein that undergoes posttranslational gamma-carboxylation and propeptide cleavage during biosynthesis. The propeptide contains the gamma-carboxylation recognition site that directs gamma-carboxylation. To identify the intracellular sites of carboxylation and propeptide cleavage, we monitored the synthesis of prothrombin in Chinese hamster ovary cells stably transfected with the prothrombin cDNA by immunofluorescent staining. The vitamin K-dependent carboxylase was located in the endoplasmic reticulum and Golgi complex. Antibodies specific to prothrombin processing intermediates were used for immunocytolocalization. Anti-des-gamma-carboxyprothrombin antibodies stained only the endoplasmic reticulum whereas antiproprothrombin antibodies (specific for the propeptide) and antiprothrombin:Mg(II) antibodies (which bind the carboxylated forms of proprothrombin and prothrombin) stained both the endoplasmic reticulum and the Golgi complex. Antiprothrombin:Ca(II)-specific antibodies (which bind only to the carboxylated form of prothrombin lacking the propeptide) stained only the Golgi complex and secretory vesicles, and colocalized with antimannosidase II and anti-p200 in the juxtanuclear Golgi complex. These results indicate that uncarboxylated proprothrombin undergoes complete gamma-carboxylation in the endoplasmic reticulum and that gamma-carboxylation precedes propeptide cleavage during prothrombin biosynthesis.

Construction, expression, and immunogenicity of chimeric HIV-1 virus-like particles.

The group-specific antigens Pr55gag of human immunodeficiency virus type-1 (HIV-1) self-assemble into noninfectious virus-like particles (VLP) that are released from various eucaryotic cells by budding. Deletion analysis of Pr55gag mutants revealed three domains into which sequences of the third variable domain V3 or the CD4-binding domain of the gp120 external glycoprotein can be inserted without destroying the capacity of the chimeric proteins to assemble to VLP. Immunization of rabbits with different types of purified chimeric VLP without adjuvants raised a strong antibody response to the Pr55gag carrier component. The magnitude of the antibody response to the inserted gp 120 epitopes strictly depended on their position within the gag polyprotein. These antisera exhibited only weak neutralizing activity. However, BALB/c mice immunized by different routes with different types of chimeric Pr55gag/V3 VLP without adjuvants developed a strong MHC class I (Dd)-restricted, cytolytic CD8+ T-cell (CTL) reactivity against a known epitope within the V3 domain. When the recombinant antigen was emulsified in mineral oil (incomplete Freund's adjuvant) or adsorbed in aluminium hydroxide, its immunogenicity for CTL was drastically reduced or completely abrogated. The magnitude of the V3-specific CTL response was not influenced by the position of the V3 domain within the Pr55gag-carrier moiety; the flanking residues, hence, did not influence processing of the exogenous antigen for MHC class I-restricted peptide presentation. These results indicate ways for the rational design and optimal delivery of CTL-stimulating HIV candidate vaccines.

Trichohyalin expression in skin tumors: retrieval of trichohyalin antigenicity in tissues by microwave irradiation.

BACKGROUND: The antitrichohyalin antibody AE 15 is effective for identifying the cell lineage that undergoes the pathway of inner root sheath-type differentiation. Unfortunately, the AE 15 does not react with trichohyalin in tissue that is formalin-fixed and embedded in paraffin according to routine procedures. METHODS: We attempted to retrieve the trichohyalin antigenicity in formalin-fixed, paraffin-embedded biopsy specimens that included normal skin as well as skin tumors such as trichofolliculoma and pilotricoma. RESULTS: We found that the use of a metal solution in combination with microwave oven heating improves the trichohyalin immunoreactivity substantially. Further, trichohyalin was found to be expressed not only in the secondary hair structure in trichofolliculoma but also in a certain cell lineage that differentiates to squamoid cells in pilomatricoma. CONCLUSIONS: Our findings established that surgical specimens processed under routine procedures can be successfully investigated with AE 15 using the microwave irradiation method. Studies of epidermal diseases expressing trichohyalin should provide valuable insights into our understanding the functional significance of trichohyalin during abnormal keratinization.

Mutant vasopressin precursors in the human hypothalamus: evidence for neuronal somatic mutations in man.

We report here the expression of mutant proteins displaying the +1 reading frame of the vasopressin and oxytocin precursors in magnocellular neurons of the human hypothalamus. Our data demonstrate a high frequency of frameshift mutations in these neurons and thus provide the first evidence of somatic mutations in neurons of the human brain. The results imply that other neuronal populations and specific genes may also undergo similar mutational events with possible consequences for neuronal functioning and pathology.

Generation and characterization of an antiserum reactive with a proteolytic processing site within rat procorticotrophin-releasing hormone.

In this paper we report the generation of an antibody specific for the cleavage site within procorticotrophin-releasing hormone (proCRH) at the N-terminus proCRH/CRH (1-41) junction. Using radioimmunoassay techniques were show that the antibody generated (781) cross-reacts specifically with the proCRH (137-150) Tyr fragment, corresponding to the cleavage site within the full length precursor molecule. The anti-cleavage site antibody does not crossreact with the endoproteolytic products originated from the CRH precursor molecule, i.e. CRH (1-41) or proCRH (125-151) or with any of the CRH-immunoreactive fragments tested i.e. CRH (36-41), CRH (1-20) and CRH (30-41). It also shows no cross-reactivity with CRH-related substances from other species, i.e. urotensin I (fish) and sauvagine (frog). The cleavage site antibody (781), recognizes the full length proCRH molecule in Western blotting and in liquid phase radioimmunoassay from transfected CHO-K1 cells expressing the full length pre-proCRH cDNA. Using immunofluorescence and immunoprecipitation techniques followed by SDS-PAGE and autoradiography, we confirm the presence of the intact CRH precursor molecule within the nucleus and the cytoplasm of stably transfected CHO-K1 cells expressing immunoreactive proCRH. The immunofluorescence studies using primary cultures of hypothalamic neurons, show that immunoreactive (IR) proCRH is localized within the perinuclear region and was also seen along the neuronal processes where it accumulates at their tips. Our results, therefore, show that this antibody will be an invaluable tool in the study of intracellular trafficking in relation to the endoproteolytic processing of the CRH precursor molecule.