Assessment of the role of intracloacal inoculation of live infectious bursal disease vaccine in breaking through maternally derived antibodies.

Infectious bursal disease (IBD) remains a potential worldwide threat to the poultry industry despite several vaccination approaches. Because maternally derived antibodies (MDA) constitute a critical problem for IBD vaccination, we examined the efficiency of the intracloacal vaccination approach in breaking through MDA. Experiment 1 determined the ability of the vaccinal strain to multiply in the bursa of Fabricius (BF) in chicks with a high level of MDA. Using real-time polymerase chain reaction, we quantified the strain in the bursae of vaccinated and non-vaccinated chicks. Experiment 2 was performed on three groups of chicks with high levels of MDA: group 1, non-vaccinated non-challenged; group 2, non-vaccinated challenged; and group 3, vaccinated challenged. Seroconversion to IBDV was measured using enzyme-linked immunosorbent assay. Groups 2 and 3 were challenged by vvIBDV at 25 days of age. Experiment 3 studied the effect of early IBD vaccinal strain multiplication on the immune response of vaccinated and non-vaccinated chicks to other vaccines. In experiment 1, the vaccinal strain showed progressive multiplication and reached the detectable titre in BF at 12 h post-vaccination despite high MDA titre. Experiment 2 showed that chicks in group 3 had significant seroconversion against IBDV. After challenge, group 3 showed significant improvements in several measured parameters compared with group 2. Moreover, results of experiment 3 proved that early multiplication of the vaccinal strain in the BF has no significant effect on the immune system or immune response to other vaccines. These results proved the promising success of this IBD vaccination approach.RESEARCH HIGHLIGHTS IBD vaccinal strain succeeded in multiplying in BF after intracloacal inoculation.Vaccinated chicks showed significant seroconversion of IBDV antibody titres.Vaccinated chicks showed a significant protection level against vvIBDV.Early IBD vaccination did not affect the immune response to other vaccines.

Synergistic induction of apoptosis and chemosensitization of human colorectal cancer cells by histone deacetylase inhibitor, scriptaid, and proteasome inhibitors: potential mechanisms of action.

Histone deacetylase inhibitors (HDACIs) exhibit modest results as single agents in preclinical and clinical studies against solid tumors; they often fall short and activate nuclear factor kappa-B (NFκB). Co-administration of HDACI with proteasome inhibitors (PIs), which interrupt NFκB pathways, may enhance HDACI-lethality. The goal of this study was to determine whether PIs could potentiate HDACI, scriptaid (SCP)-mediated lethality, to unravel the associated mechanisms and to assess the effects of the combined inhibition of HDAC and proteasome on chemotherapy response in human colorectal cancer cells. Cancer cells were exposed to agents alone or in combination; cell growth inhibition was determined by MTT and colony formation assays. HDAC-, proteasome-, NFκB-activities, and reactive oxygen species (ROS) were quantified. Induction of apoptosis and cell cycle alterations were monitored by flow cytometry. Expression of cell cycle/apoptosis and cytoprotective/stress-related genes was determined by real-time qRT-PCR and EIA, respectively. Potentiation of cancer cell sensitivity to chemotherapies by SCP/PIs was also evaluated. SCP and PIs: MG132, PI-1, or epoxomicin interact synergistically to potently inhibit cancer cell growth, alter cell cycle, induce apoptosis, reduce NFκB activity, and increase ROS generation. These events are associated with multiple perturbations in the expression of cell cycle, apoptosis, cytoprotective, and stress-related genes. Co-administration of SCP and PIs strikingly increases the chemosensitivity of cancer cells (122-2 × 10(5)-fold) in a drug and SCP/PIs-dependent manner. This combination regimen markedly reduced the doses of chemotherapies with potent anticancer effects and less toxicity. A strategy combining HDAC/proteasome inhibition with chemotherapies warrants further investigation in colorectal cancer.

Effects of timing and dose of vitamin A on tissue retinol concentrations and antibody production in the previously vitamin A-depleted rats.

The effect of retinol repletion in previously vitamin A-depleted Lewis rats on antibody production to pneumococcal polysaccharide (SSS-III) was studied. When vitamin A-depleted rats were given either 0.35 mumol (0.1 mg) or 5.2 mumol (1.5 mg) retinol, plasma retinol became normal within 8 h. Liver and lymphoid-organ retinol concentrations were normalized by 1 d after repletion with 5.2 mumol but not 0.35 mumol retinol. Antibody production to SSS-III was compared after administering 5.2 mumol retinol either as a divided dose (half given 4 d before and half given on the day of immunization) or as a single dose concurrent with immunization. Vitamin A-depleted rats produced very little SSS-III-specific antibody. The divided dose of retinol consistently restored anti-SSS-III production whereas the single concurrent dose was less effective despite equal effects on tissue retinol concentrations. Interestingly, normalization of plasma retinol was not always a good predictor of the immune response to pneumococcal polysaccharide.

Human glioma associated intermediate filament proteins: over-expression, co-localization and cross-reactivity.

The identification of human brain tumor-associated markers could facilitate the development of new diagnostic and therapeutic strategies for these malignancies. The type III intermediate filament proteins (IFPs): vimentin, desmin and glial fibrillary acidic protein (GFAP) were studied in human glioma tissue extracts, in sera from glioma patients and in low passage glioma cell lines prepared from primary cultures of freshly dissected tumors. Radioimmunoassay (RIA) studies, using anti-GFAP, anti-desmin and anti-vimentin mAbs, showed high levels of these proteins in glioma extracts. Binding studies with authentic IFPs indicated the absence of circulating antibodies against these proteins in the sera of glioma patients. On the other hand, these sera showed high levels of vimentin. Binding studies with these antibodies using RIAs and western immunoblotting, showed that while anti-GFAF mAbs were specific to GFAP, anti-desmin mAb cross-reacted completely with GFAP, anti-vimentin mAb cross-reacted substantially with desmin and GFAP. Immunofluorescence staining of frozen sections revealed high levels of neurofilaments in gliomas and strikingly low levels in normal brain tissue. Double immunofluorescence staining showed co-occurrence of all three IFPs in the same filaments. This suggests either co-expression or cross-reactivity of these proteins due to their high degree of homology. Thus, caution should be exercised in the use and interpretation of immunohistochemical data using antibodies to IFs.

Growth rate characteristics of acoustic neuromas associated with neurofibromatosis type 2.

Neurofibromatosis type 2 (NF2) is a dominantly inherited disorder characterized by the occurrence of bilateral acoustic neuromas (ANs and other central nervous system tumors. Magnetic resonance images and audiologic data on 22 patients with NF2 who underwent multiple studies at the National Institutes of Health between 1983 and 1993 were reviewed to determine the growth characteristics of ANs in these patients. The average growth rate of ANs in NF2 patients was 0.30 cm3 per year and was significantly higher in older patients (0.75 cm3 per year) than in younger ones (0.12 cm3 per year). Larger ANs were more commonly found in patients with concomitant spinal tumors or meningiomas. NF2 patients with spinal tumors but not meningiomas demonstrated faster growth rates than patients without additional tumor burden. The data from this study suggest that older patients or patients with associated spinal tumors have faster growing ANs and therefore should be followed closely and treated aggressively.

Impairment, disability, and other medical-legal aspects of dysphonia.

As awareness of the importance of the human voice has grown, so too have legal issues surrounding voice dysfunction. Otolaryngologists must be familiar with principles of impairment and disability and with appropriate medical-legal management for voice patients.

Laryngeal manifestations of postpoliomyelitis syndrome.

Postpoliomyelitis syndrome (PPS) is a disease that may occur in survivors of acute poliomyelitis several decades after their initial infection. It can present as dysphonia, with vocal weakness and fatigue. Swallowing, respiratory, and other laryngopharyngeal symptoms may be manifestations of the disease or they may represent worsening of previously stable and compensated deficits. Three cases of laryngeal changes in PPS with videostroboscopic and laryngeal electromyography findings highlight the features of this disorder. We review possible etiologies of laryngeal PPS, diagnostic criteria, and treatment, as well as the current literature.

Management of vocal fold scar with autologous fat implantation: perceptual results.

Vocal fold scar disrupts the mucosal wave and interferes with glottic closure. Treatment involves a multidisciplinary approach that includes voice therapy, medical management, and sometimes surgery. We reviewed the records of the first eight patients who underwent autologous fat implantation for vocal fold scar. Information on the etiology of scar, physical findings, and prior interventions were collected. Videotapes of videostroboscopic findings and perceptual voice ratings [Grade, Roughness, Breathiness, Asthenia, Strain (GRBAS)] were randomized and analyzed independently by four blinded observers. Etiology of scar included mass excision (7), vocal fold stripping (3), congenital sulcus (2), and hemorrhage (1). Prior surgical procedures performed included thyroplasty (1), autologous fat injection (9), excision of scar (2), and lysis of adhesions (2). Strobovideolaryngoscopy: Statistically significant improvement was found in glottic closure, mucosal wave, and stiffness (P = 0.05). Perceptual ratings (GRBAS): Statistically significant improvement was found in all five parameters, including overall Grade, Roughness, Breathiness, Asthenia, and Strain (P = 0.05). Patients appear to have improved vocal fold function and quality of voice after autologous fat implantation in the vocal fold. Autologous fat implantation is an important adjunctive procedure in the management of vocal fold scar, and a useful addition to the armamentarium of the experienced phonomicrosurgeon.

Laryngeal myasthenia gravis: report of 40 cases.

Myasthenia gravis, an autoimmune disorder of the neuromuscular junction, is usually recognized because of ocular complaints or generalized weakness. We report a series of 40 patients who presented with dysphonia as their initial and primary complaint. Diagnostic testing included strobovideolaryngoscopy, electromyography (EMG) with repetitive stimulation and Tensilon testing, and laboratory and radiographic evaluation. Strobovideolaryngoscopy most commonly revealed fluctuating impairment of vocal fold mobility, either unilateral or bilateral. EMG detected evidence of neuromuscular junction abnormalities in all patients. Only one patient had evidence of antiacetylcholine receptor (ACh-R) antibodies, but many other abnormalities suggestive of autoimmune dysfunction were present. Pyridostigmine therapy was initiated in 34 patients but was not tolerated in 4. Of the remaining 30 patients, 23 reported improvement of symptoms. We conclude that myasthenia gravis can present with symptoms confined primarily to the larynx and should be included in the differential diagnosis of dysphonia.

Psychiatric manifestations of medications commonly prescribed in otolaryngology.

Otolaryngologists, nurses, and psychological professionals should be familiar with the potential psychiatric side effects of medications that are commonly prescribed by otolaryngologists. Because some of these side effects are atypical, their relationship to medications might not be obvious. An awareness of the potential for psychiatric side effects caused by adrenocorticoids, antihistamines and decongestants, and antisecretory medications will help the clinician avoid or detect and treat drug-induced disorders, as will an awareness of the potential for side effects caused by combinations of medications. Identification of individual risk factors such as age, pre-existing organic brain disease, a history of drug abuse or dependence, or coexisting or pre-existing psychiatric disorders is important in preventing and detecting drug-induced psychiatric disorders. The drugs discussed in this article can have serious, even fatal, interactions with certain psychiatric medications.

Effects of amino acid substitutions outside an antigenic site on protein binding to monoclonal antibodies of predetermined specificity obtained by peptide immunization: demonstration with region 145-151 (antigenic site 5) of myoglobin.

Monoclonal antibodies of predetermined specificity were prepared by immunization with a free (i.e., without coupling to any protein carrier) synthetic peptide representing region 145-151 of sperm whale myoglobin (SpMb) and their cross-reactions with eight Mb variants were determined. Five Mbs--bottle-nose dolphin myoglobin (BdMb), pacific common dolphin myoglobin (PdMb), horse myoglobin (HsMb), dog myoglobin (DgMb), and badger myoglobin (BgMb)--have an identical sequence in that region. Nevertheless, these Mbs exhibited very different cross-reactivities. BdMb and PdMb exhibited cross-activities which were comparable to that of the reference antigen, SpMb; while the reactivity of HsMb was remarkedly decreased, DgMb and BgMb showed almost no cross-reactions with these mAbs. Since the region 145-151 has an identical sequence in all the five Mbs, it is concluded that the differences in their antigenic reactivities with anti-region 145-151 mAbs are due to the effects of amino acid substitutions outside the region 145-151. Another pair of myoglobins, echidna myoglobin (EdMb) and chicken myoglobin (ChMb), have the same sequence in that region, but reacted very differently with anti-region 145-151 mAbs. The reactivity and affinity of EdMb were substantially decreased while those of ChMb were almost completely absent, relative to SpMb. It is concluded, contrary to popular assumptions, that when an amino acid substitution influences the binding of a protein variant to a mAb, it is not necessary for that substitution to be an actual contact residue (i.e., a residue within the antigenic site where the mAb binds). Such effects, which are often very drastic, could be due to indirect influences of the substitution on the chemical and binding properties of the site residues. Furthermore, residues which had been postulated, on the basis of these assumptions, to constitute discontinuous antigenic sites in SpMb, were found [from the present studies and those recently reported with mAbs against the other four antigenic site of Mb (regions 15-22, 56-62, 94-100, and 113-120 of SpMb)] to merely be exerting indirect effects on the known five antigenic sites of Mb. The effects of substitutions, which can happen even in the absence of conformational changes, are determined by many factors, such as the chemical nature of the substitution, its environment, its distance from the site, and the nature of the site residue(s) being affected.

Effects of amino acid substitutions outside an antigenic site on protein binding to monoclonal antibodies of predetermined specificity obtained by peptide immunization: demonstration with region 113-120 (antigenic site 4) of myoglobin.

Immunochemical cross-reactivity of protein variants has been very frequently used to map protein antigenic sites. The approach is based on the assumption that amino acid substitutions affecting the binding of a protein to its antibody, particularly when monoclonal antibodies (mAbs) are used, must be part of the antigenic site and not far from it. The assumption was investigated in this study by determining the effects of amino acid substitutions outside the antigenic site on the reactivity of six myoglobin (Mb) variants with three mAbs of predetermined specificity prepared by immunization with a free synthetic peptide representing region 113-120 (antigenic site 4) of Mb. Two of the Mb variants used had no substitutions within residues 113-120 (the region to which the specificity of the mAbs is directed) and yet exhibited markedly decreased cross-reactions and binding affinities, relative to the reference antigen, sperm-whale Mb. The other three Mb variants possessed substitutions within, as well as outside, region 113-120 and showed very little cross-reactivities. The results of this study, particularly with the Mbs that have no substitutions within the indicated antigenic site, clearly show that substitutions outside the site, and which by design are not part of the site, can influence very markedly the reactivity of the protein variant with the anti-site mAbs. The approach can, therefore, lead to serious errors if used to identify residues of protein antigenic sites.

Effects of amino acid substitutions outside an antigenic site on protein binding to monoclonal antibodies of predetermined specificity obtained by peptide immunization: demonstration with region 94-100 (antigenic site 3) of myoglobin.

Amino acid substitutions outside protein antigenic sites are very frequently assumed to exert no effect on binding to antiprotein antibodies, especially if these are monoclonal antibodies (mAbs). In fact, a very popular method for localization of residues in protein antigenic sites is based on the interpretation that whenever a replacement causes a change in binding to antibody, then that residue will be located in the antigenic site. To test this assumption, mAbs of predetermined specificity were prepared by immunization with a free (i.e., without coupling to any carrier) synthetic peptide representing region 94-100 of sperm whale myoglobin (Mb). The cross-reactivities and relative affinities of three mAbs with eight Mb variants were studied. Five Mb variants which had no substitutions within the boundaries of the designed antigenic site exhibited remarkable, and in two cases almost complete, loss in cross-reactivity relative to the reference antigen, sperm whale Mb. Two myoglobins, each of which had one substitution within region 94-100, showed little or no reactivity with the three mAbs. It is concluded that substitutions outside an antigenic site can exert drastic effects on the reactivity of a protein with mAbs against the site and that caution should be exercised in interpreting cross-reactivity data of proteins to implicate residues directly in an antigenic site.

Effects of amino acid substitutions outside an antigenic site on protein binding to monoclonal antibodies of predetermined specificity obtained by peptide immunization: demonstration with region 56-62 (antigenic site 2) of myoglobin.

This work was carried out in order to study the effects of substitutions outside antigenic site 2 of sperm whale myoglobin (SpMb) on the reactivity of protein variants with antisite 2 monoclonal antibodies (mAbs). A synthetic peptide corresponding to region 56-62 (site 2) of SpMb was used as an immunogen in mice in its free form (i.e., without coupling to any carrier) to prepare a panel of mAbs whose predetermined specificity is directed, by design, against this region. The binding of three of these mAbs to eight Mbs from different species was studied. Myoglobins of Pacific common dolphin, finback whale, and horse, which have no substitutions within region 56-62 relative to SpMb, showed remarkable differences in their cross-reactivities and relative affinities with each of the mAbs. Myoglobins of badger, chicken, and dog, although they have an identical substitution within the site (Ala-57 to Gly), exhibited cross-reactivities with a given mAb that were affected differently. Echidna Mb, which has one replacement (Glu-59 to Ala) within region 56-62, displayed greatly reduced cross-reactivities and relative binding affinities. The results, especially those from Mbs that have no substitutions within the boundaries of site 2, clearly indicate that substitutions outside site 2 of Mb can exert drastic effects on the binding of the Mb variants with mAbs whose specificity was predesigned to be against the site. These indirect effects and their impact on site reactivity will completely explain previous findings on cross-reactivities of Mb variants with mAbs of unknown specificity and will rule out the postulations of discontinuous sites in Mb, which were based on the assumption that every substitution affecting reactivity is directly involved in binding to antibody.

In vitro efficacy of anti-glial fibrillary acidic protein monoclonal antibodies against human malignant glioma cell lines.

Our studies have confirmed the presence of large concentrations of various intermediate filament proteins (IFPs) in glioma tissue compared to normal brain. This avenue of research was extended to assess the anti-proliferative activity of anti-intermediate filament protein monoclonal antibodies (anti-IFP mAbs) against human glioma cells. In this study, anti-proliferative activity of glial fibrillary acidic protein monoclonal antibodies (anti-GFAP mAbs) has been tested in vitro, using glioma cell lines prepared and established from freshly resected brain tumors. One anaplastic astrocytoma (AA), two glioblastoma multiforme (GB1 and GB2) cell lines and three anti-GFAP mAbs (B12C4, B12B4 and B6C6, all IgG1, kappa) were used. Immunofluorescence study indicated the ability of anti-GFAP mAbs to recognize the cell surface of glioma cells and the inhibition study showed that mAb B12B4 inhibited the proliferation of GB1 (96%), GB2 (85%) and AA (93%) at a concentration of 3.2 x 10(-10) M. mAb B12C4 inhibited the proliferation of GB1 (95%), GB2 (86%) and AA (94%) at a concentration of 3.26 x 10(-10) M and mAb B6C6 inhibited the proliferation of GB1 (75%), GB2 (75%) and AA (91%) at a concentration of 2.074 x 10(-10) M. Thymidine release assay demonstrated the cytolytic activities of anti-GFAP mAbs towards these glioma cell lines, and this observation was confirmed by dye exclusion, which indicated the lysis of glioma cells after anti-GFAP mAbs treatment. Anti-GFAP mAbs had little effect (< or = 20%) on normal human lymphocyte, liver and intestine cell lines. These results look promising for radioimaging and immunotherapy of human gliomas.

The effect of injectable contraceptive on lipid metabolism in women.

The effect of long acting progestational contraceptive injection, norethisterone-oenanthate was studied in twenty six women. The post injection results showed a significant decrease in total lipids, free cholesterol, total cholesterol, triglycerides, phospholipids and free fatty acids.

PIP: 26 healthy Egyptian women, aged 20-35 years, were studied to determine the effects of norethindrone-enanthate in injectable form on lipid metabolism. The dose was 200 mg, with the 1st injection being given on Day 5 of the menstrual cycle. Blood samples were taken before injection, while the subjects were in a fasting state, and 2 months after the last injection, a total of 6 months later. Injections were repeated every 2 months in the interim. Levels of total lipids, free cholesterol, total cholesterol, triglycerides, phospholipids, and free fatty acids were compared. Total serum lipids decreased significantly after 2, 4, and 6 months of injection (significantly different from pretreatment values at the level of 1%). Total and free serum cholesterol both decreased significantly after 2, 4, and 6 months of injection. Serum triglycerides and serum phospholipids were likewise significantly decreased after 2, 4, and 6 months of injection. however, plasma free fatty acids were significantly lower after 2 months but not after 4 and 6 months of injection. The decrease in free fatty acids may be attributed to suppression of lipolysis due to decreased cyclic adenosine monophosphate or to epinephrine suppression; triglyceride suppression may have resulted from a possible effect on estrogen receptors decreased transport of lowered fatty acids to adipose tissues or increased lipoprotein lipase; phospholipid reduction may be related to the lessening of fatty acids being transported to the adipose tissues or to impaired liver function; and total lipid suppresson was thought to be the result of the significant decrease in blodd lipid fractions observd. it is concluded that this injectable preparation is more satisfactory, in terms of lipid metabolic effects, than combined contraceptive preparatios.