Cheeklift With and Without Posterior Lamellar Spacer Grafts for Treatment of Lower Eyelid Retraction.

BACKGROUND: To compare outcomes of lower eyelid retraction repair using a subperiosteal midface lifting technique with and without posterior lamellar grafts. METHODS: Charts of patients undergoing a sub-periosteal midface lift for treatment of lower eyelid retraction using 4 techniques for posterior lamellar reconstruction were reviewed. Thirty patients were included in each of the groups: midface with hard palate graft (HPG), midface lift with acellular cadaveric graft (ADG), midface lift with retractor disinsertion (RD) and midface lift alone (NG). Measurements of distance from pupil center to lower lid margin (MRD2) and from lateral limbus to lower lid margin (MRD2limbus) were taken from pre- and postoperative photographs and compared. Secondary outcomes included rates of reoperation, major and minor complications, resolution of symptoms and keratopathy. RESULTS: One hundred twenty operations were assessed (n = 30 for each surgical group). The average follow-up time was 20 weeks. The median MRD2 elevation was 0.95 mm (NG), 0.85 mm (HPG), 1.59 mm (ADG) and 1.02 mm (RD). The median MRD2limbus elevation was 1.06 mm (NG), 0.92 mm (HPG), 1.45 mm (ADG) and 1.12 mm (RD). There were no significant differences in MRD2 or MRD2limbus between the 4 groups (p = 0.06 and 0.29, respectively). Reoperation rates were highest with in the hard palate graft group (33%) compared to other techniques (p = 0.0006). CONCLUSIONS: Similar degrees of lower eyelid elevation were achieved with all the midface lifting techniques, and complication rates did not significantly differ between techniques. However, the higher reoperation rates with the use of spacer grafts suggest that a no-graft technique may be preferable. LEVEL OF EVIDENCE III: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

The enteroinsular axis during hospitalization in newborn foals.

The enteroinsular axis (EIA) is an energy regulatory system that modulates insulin secretion through the release of enteroendocrine factors (incretins). Despite the importance of energy homeostasis in the equine neonate, information on the EIA in hospitalized foals is lacking. The goals of this study were to measure serum insulin and plasma incretin (glucose-dependent insulinotropic polypeptide [GIP], glucagon-like peptide-1 [GLP-1] and glucagon-like peptide-2 [GLP-2]) concentrations, to determine the insulin and incretin association, as well as their link to disease severity and outcome in hospitalized foals. A total of 102 newborn foals ≤72 h old were classified into hospitalized (n = 88) and healthy groups (n = 14). Hospitalized foals included septic (n = 55) and sick non-septic (SNS; n = 33) foals based on sepsis scores. Blood samples were collected over 72 h to measure serum insulin and plasma GIP, GLP-1 and GLP-2 concentrations using immunoassays. Data were analyzed by nonparametric methods and univariate logistic regression. At admission, serum glucose and insulin and plasma GIP were significantly lower in hospitalized and septic compared to healthy foals (P < 0.01), while plasma GLP-1 and GLP-2 concentrations were higher in hospitalized and septic foals than healthy and SNS foals, and decreased over time in septic foals (P < 0.05). As a percent of admission values, GLP-1 and GLP-2 concentrations dropped faster in healthy compared to hospitalized foals. Serum insulin concentrations were lower in hospitalized and septic non-survivors than survivors at admission (P < 0.01). Hospitalized foals with serum insulin < 5.8 µIU/mL, plasma GLP-1 >68.5 pM, and plasma GLP-2 >9 ng/mL within 24 h of admission were more likely to die (OR = 4.2; 95% CI = 1.1-16.1; OR = 13.5, 95% CI = 1.4-123.7; OR = 12.5, 95% CI = 1.6-97.6, respectively; P < 0.05). Low GIP together with increased GLP-1 and GLP-2 concentrations indicates that different mechanisms may be contributing to reduced insulin secretion in critically ill foals, including impaired intestinal production (GIP, proximal intestine) and pancreatic endocrine resistance to enhanced incretin secretion (GLP-1, GLP-2; distal intestine). These imbalances could contribute to energy dysregulation in the critically ill equine neonate.

CRISPR/Cas9 gene editing demonstrates metabolic importance of GPR55 in the modulation of GIP release and pancreatic beta cell function.

G-protein coupled receptor-55 (GPR55), an endocannabinoid receptor, is a novel anti-diabetic target. This study aimed to assess the metabolic functionality of GPR55 ligands using CRISPR/Cas9 gene editing to determine their regulatory role in beta cell function and incretin-secreting enteroendocrine cells. A clonal Gpr55 knockout beta cell line was generated by CRISPR/Cas9 gene editing to investigate insulin secretion and Gpr55 signalling. Acute effects of GPR55 agonists were investigated in high fat fed (HFD) diabetic HsdOla:TO (Swiss TO) mice. Atypical and endogenous endocannabinoid ligands (10-7-10-4M) stimulated insulin secretion (p < 0.05-0.001) in rodent (BRIN-BD11) and human (1.1B4) beta cells, with 2-2.7-fold (p < 0.001) increase demonstrated in BRIN-BD11 cells (10-4M). The insulinotropic effect of Abn-CBD (42 %), AM251 (30 %) and PEA (53 %) were impaired (p < 0.05) in Gpr55 knockout BRIN-BD11 cells, with the secretory effect of O-1602 completely abolished (p < 0.001). Gpr55 ablation abolished the release of intracellular Ca2+ upon treatment with O-1602, Abn-CBD and PEA. Upregulation of insulin mRNA by Abn-CBD and AM251 (1.7-3-fold; p < 0.01) was greatly diminished (p < 0.001) in Gpr55 null cells. Orally administered Abn-CBD and AM251 (0.1 µmol/kgBW) improved GIP (p < 0.05-p < 0.01), GLP-1 (p < 0.05-p < 0.001), glucose tolerance (p < 0.001) and circulating insulin (p < 0.05-p < 0.001) in HFD diabetic mice. Abn-CBD in combination therapy with DPP-IV inhibitor (Sitagliptin) resulted in greater improvement in glucose tolerance (p < 0.05) and insulin release (p < 0.05). Antagonism of Gpr55 in-vivo attenuated the glucoregulatory effects of Abn-CBD (p < 0.05). Conclusively, GPR55 agonists enhance insulin, GIP and GLP-1 release, thereby promoting GPR55 agonist monotherapy and combinational therapy as a novel approach for the treatment of type-2-diabetes.

Capsid Engineering Overcomes Barriers Toward Adeno-Associated Virus Vector-Mediated Transduction of Endothelial Cells.

Endothelial cells (EC) are targets in gene therapy and regenerative medicine, but they are inefficiently transduced with adeno-associated virus (AAV) vectors of various serotypes. To identify barriers hampering efficient transduction and to develop an optimized AAV variant for EC transduction, we screened an AAV serotype 2-based peptide display library on primary human macrovascular EC. Using a new high-throughput selection and monitoring protocol, we identified a capsid variant, AAV-VEC, which outperformed the parental serotype as well as first-generation targeting vectors in EC transduction. AAV vector uptake was improved, resulting in significantly higher transgene expression levels from single-stranded vector genomes detectable within a few hours post-transduction. Notably, AAV-VEC transduced not only proliferating EC but also quiescent EC, although higher particle-per-cell ratios had to be applied. Also, induced pluripotent stem cell-derived endothelial progenitor cells, a novel tool in regenerative medicine and gene therapy, were highly susceptible toward AAV-VEC transduction. Thus, overcoming barriers by capsid engineering significantly expands the AAV tool kit for a wide range of applications targeting EC.

Gene editing in the context of an increasingly complex genome.

The reporting of the first draft of the human genome in 2000 brought with it much hope for the future in what was felt as a paradigm shift toward improved health outcomes. Indeed, we have now mapped the majority of variation across human populations with landmark projects such as 1000 Genomes; in cancer, we have catalogued mutations across the primary carcinomas; whilst, for other diseases, we have identified the genetic variants with strongest association. Despite this, we are still awaiting the genetic revolution in healthcare to materialise and translate itself into the health benefits for which we had hoped. A major problem we face relates to our underestimation of the complexity of the genome, and that of biological mechanisms, generally. Fixation on DNA sequence alone and a 'rigid' mode of thinking about the genome has meant that the folding and structure of the DNA molecule -and how these relate to regulation- have been underappreciated. Projects like ENCODE have additionally taught us that regulation at the level of RNA is just as important as that at the spatiotemporal level of chromatin.In this review, we chart the course of the major advances in the biomedical sciences in the era pre- and post the release of the first draft sequence of the human genome, taking a focus on technology and how its development has influenced these. We additionally focus on gene editing via CRISPR/Cas9 as a key technique, in particular its use in the context of complex biological mechanisms. Our aim is to shift the mode of thinking about the genome to that which encompasses a greater appreciation of the folding of the DNA molecule, DNA- RNA/protein interactions, and how these regulate expression and elaborate disease mechanisms.Through the composition of our work, we recognise that technological improvement is conducive to a greater understanding of biological processes and life within the cell. We believe we now have the technology at our disposal that permits a better understanding of disease mechanisms, achievable through integrative data analyses. Finally, only with greater understanding of disease mechanisms can techniques such as gene editing be faithfully conducted.

A survey of patients with substance use disorders at government treatment facilities in The Bahamas / Estudio de pacientes con trastornos por uso de sustancias en centros gubernamentales de tratamiento en Las Bahamas

OBJECTIVE: To determine the sociodemographic information and characteristics of patients aged 18-60 years diagnosed with substance use disorders presenting to the three government treatment facilities. To determine the prevalence rates of alcohol, cannabis, cocaine and poly-substance use disorders in patients presenting to government treatment facilities. METHODS: The Structured Clinical Interview for Diagnostic and Statistical Manual of Mental Disorders fourth edition, text revision (DSM-IV-TR) Axis 1 disorders was the first instrument used to screen for drug abuse or dependence. Additional questionnaires included a sociodemographic questionnaire and the Survey of Addicted Patients in Treatment Centre Standardized Questionnaire. RESULTS: The number of participants interviewed in the study was 120 people; 89.2% were males and 10.8% were females. The mean age of all participants with substance use disorders was 36.22 (10.74) years and they were predominantly male (8 to 1). Males were mostly single, unemployed or casually employed, of middle school education and were residents of New Providence. Alcohol, cannabis and cocaine were the common drugs that were misused. Of cocaine users, 52 (82.5%) met the DSM-IV-TR criteria for dependence and of cannabis users, 20 (18.9%) met the DSM-IV-TR criteria for abuse. CONCLUSIONS: There is a need to conduct community surveys on school children, other adult populations eg in the wider community and on other island populations to determine the population rates of substance use disorders. Once the needs have been identified through research for the different islands and target groups, informed decisions can be made as to the allocation of financial and human resources.

OBJETIVO: Determinar la información sociodemográfica y las características de los pacientes en edades de 18-60 años diagnosticadas con trastornos por uso de sustancia, que acuden a los tres centros gubernamentales de tratamiento de la drogadicción. Determinar la tasa de prevalencia de los trastornos por uso de alcohol, cannabis, cocaína y polisustancias en los pacientes que acuden a los centros de tratamiento del gobierno. MÉTODOS: La Entrevista clínica estructurada para el diagnóstico y el Manual estadístico de trastornos mentales, cuarta edición, texto revisado (DSM-IV-TR), trastornos del eje 1, fue el primer instrumento utilizado para detectar el abuso o dependencia de drogas. Los cuestionarios adicionales incluyen un cuestionario sociodemográfico así como la llamada Encuesta de pacientes adictos en el cuestionario estandarizado de los centros de tratamiento. RESULTADOS: El número de participantes entrevistados en el estudio fue de 120 personas; 89.2% eran varones y 10,8% eran hembras. La edad promedio de todos los participantes con trastornos por uso de sustancias fue 36.22 (10,74) años y eran predominantemente masculinos (8 a 1). Los varones eran en su mayoría solteros, desempleados, o trabajadores eventuales, de nivel educacional medio, y residentes de Nueva Providencia. Alcohol, cannabis y cocaína fueron las comúnmente las sustancias del uso adictivo. De los consumidores de cocaína, 52 (82,5%) correspondían a los criterios del DSM-IV-TR con respecto a la dependencia, y de los consumidores de cannabis, 20 (18,9%) correspondían a los criterios de DSM-IV-TR en relación con el abuso de sustancias. CONCLUSIONES: Es necesario llevar a cabo encuestas comunitarias con niños en edad escolar, otras poblaciones adultas - por ejemplo en la comunidad en general y en otras poblaciones de la isla - para determinar las tasas poblacionales de trastornos por uso de sustancias. Una vez que las necesidades hayan sido identificadas mediante investigación de las diferentes islas y los grupos seleccionados como objetivos, pueden tomarse decisiones informadas en cuanto a la asignación de las finanzas y recursos humanos.

Peripheral axon regrowth: new molecular approaches.

Peripheral nerves, essential connections between the brain, spinal cord and body, do not regenerate as well as generally reported. Identifying new strategies to facilitate regeneration is essential to reversing neurological deficits from nerve injuries or disease. This review will discuss several selected and novel molecular insights into peripheral nerve trunk repair and axon regrowth that have the potential to improve regenerative success. Of particular interest is the phosphatidylinositol 3-kinase (PI3K)-Akt pathway in peripheral neurons, inhibited by the constitutively expressed phosphatase tumor suppressor PTEN. Knockdown or inhibition of PTEN is associated with robust sprouting of adult sensory neurons in vitro and in vivo, additive to the accelerated outgrowth offered by the preconditioning effect. This sprouting response, if spatially and temporally constrained, may provide potent regrowth initiation, of interest in otherwise untreatable nerve damage.

Micronuclei in lymphocytes of prostate cancer patients undergoing radiation therapy.

To further verify the applicability of the micronucleus (MN) assay in biodosimetry, we measured the MN yield in cytokinesis-blocked (CB) peripheral blood lymphocytes (PBL) of eight prostate cancer (PC) patients. These patients had no previous chemotherapy or radiotherapy (xRT). They were treated with standardized schemes of fractionated pelvic xRT. Before xRT, and at one random time-point during the course of xRT, blood samples were collected from each patient for the following purposes: (1) to verify the relationship between the MN yield in PBL and the estimated equivalent (EQ) total-body absorbed dose; and (2) to evaluate the individual differences of ex vivo radiation dose-response (1-4 Gy) relationship of MN yield in PBL before xRT. The number of xRT fractions, cumulative tumor dose, and EQ total-body absorbed doses of these patients represented a wide range. We found in PBL of these patients that (1) MN yield (Y) increased linearly with the estimated EQ total-body absorbed dose as Y=14.6+9.2D (R(2)=0.7, p=0.007); the distributions of MN yield were overdispersed; the ratio of relative increment of MN yield per 1000 binucleated (BN) PBL ranged from 0.9 to 8.2 (median: 4.1) folds above that of the respective baseline levels; and (2) before xRT, the MN yields also increased linearly with the ex vivo radiation dose; at each radiation dose level, the distributions of MN yield were overdispersed in most patients. In two of the three patients with xRT-induced early side effects (cystitis, diarrhea), the MN yield in PBL induced by ex vivo irradiation before xRT was significantly higher than in the other patients without xRT-induced side effects. These findings suggest that MN yields in CB PBL can be used as an in vivo biodosimeter. Since the differences in individual ex vivo radiation dose-response relationship of MN yield in PBL before xRT appeared to be significant, our preliminary results also suggest that it may be possible to identify individual intrinsic radiosensitivity before the start of xRT.

Merkel cells in the human oesophagus.

Merkel cells (MCs) are well recognized in the basal layers of the skin and oral mucosa, but this paper describes for the first time the presence of MCs in the human oesophagus. These cells are not identified in neonatal oesophagus, but are seen singly and in clusters in adult specimens. Application of stereological techniques shows that MCs are more numerous in the mid-oesophageal region. Cells expressing established markers of MCs have also been demonstrated in two out of six primary small cell carcinomas of the oesophagus. Further investigation of the role of MCs in oesophageal innervation and epithelial biology will be of interest.

Effect of blood storage on radiation-induced micronuclei in human lymphocytes.

To evaluate the effect of blood storage on the yield of micronuclei (MN) in both irradiated (in vivo and ex vivo) and unirradiated peripheral blood lymphocytes (PBL), we applied the MN assay in cytokinesis-blocked (CB) PBL obtained from healthy subjects (n=11), and from cancer patients (n=10) who were undergoing fractionated partial-body radiotherapy (xRT). The heparinized blood samples were exposed to 137Cs-irradiation (0 Gy or 2 Gy) immediately after blood collection and were stored upright in test tubes either at room temperature (22 degrees C) or in the refrigerator (5 degrees C). Duplicate whole blood cultures from each sample were set up at 0 h, 96 h, and 120 h after ex vivo irradiation. Giemsa (10%) stained slides were prepared from each culture. MN yield was determined per 1000 binucleated cells. As compared to that obtained from the corresponding fresh blood samples, we found that (1) the 22 degrees C blood storage temperature did not affect MN yields in PBL of either healthy subjects or cancer patients up to 96 h, either with or without ex vivo irradiation; and (2) while blood samples were stored at 5 degrees C, the MN yield increased significantly in PBL of healthy subjects (with or without ex vivo irradiation) at 120 h, and in cancer patients (with ex vivo irradiation) at 96 h and 120 h. Since handling of the blood sample is important for CBMN assay during shipment or in the laboratory, our findings showed that blood storage at 22 degrees C or at 5 degrees C up to 96 h appeared to provide insignificant variations of the MN results as compared to fresh blood samples. However, the 96 h of blood storage at 5 degrees C elevated the MN frequency in ex vivo irradiated PBL of cancer patients who were undergoing xRT.

Effect of ex vivo hyperthermia on radiation-induced micronuclei in lymphocytes of cancer patients before and during radiotherapy.

To investigate the effect of ex vivo hyperthermia (HT) and 137Cs-irradiation on micronucleus (MN) production in cytokinesis-blocked lymphocytes, we obtained the peripheral blood samples from the same cancer patients (n=6) before and during fractionated partial-body radiotherapy (xRT). The whole blood cultures were heated at 43.5 degrees C for 60 min, followed by 137Cs irradiation (0-4 Gy). The control cultures from the same patients were incubated at 37 degreesC after being exposed to radiation. The lymphocytes were then stimulated with PHA. Cytochalasin B was applied at 44 h, and lymphocytes were harvested at 72 h. MN frequency was determined on Giemsa-stained slides. We found that in patients before xRT, HT (43.5 degrees C) significantly increased the MN yield (mean+/-SEM) in unirradiated lymphocytes from 15.6+/-2.8 (37 degrees C) to 39.7+/-10.9. Further, in patients either before or during xRT, when the lymphocytes were treated with HT (43.5 degrees C) and combined with ex vivo irradiation, the MN yield (Y) could be estimated by a linear equation Y=C+alphaD. Our findings indicate that as measured by the MN production in cytokinesis-blocked lymphocytes, HT alone at 43.5 degrees C++ induced DNA damage. Moreover, it enhanced the radiation-induced cytogenetic damage. Therefore, the application of HT may impair the T-cell function in cancer patients who are receiving radiotherapy. 1998 Elsevier Science B.V.

A comparison of membrane enzymes of human and pig oesophagus; the pig oesophagus is a good model for studies of the gullet in man.

The distribution and relative catalytic activities of five plasma membrane enzymes (alkaline phosphatase, dipeptidyl peptidase IV, gamma-glutamyl transpeptidase, microsomal alanyl aminopeptidase and glutamyl aminopeptidase) were examined in human and pig oesophagus. In both species, alkaline phosphatase activity occurred in basal and suprabasal cells of the epithelium and in capillaries. Stromal cells in the human submucosa were particularly reactive. Dipeptidyl peptidase IV was present in blood vessels and capillaries in man and pig and in submucous glands in the pig. The enzyme was also present in both species in the lamina propria cells immediately adjacent to the epithelial basal lamina. In the human, gamma-glutamyl transpeptidase occurred in the epithelial basal cells and in isolated basal and lower prickle cells in the pig. Stromal cells in the human submucosa were strongly reactive and capillaries in the muscularis propria in both species moderately active. Microsomal alanyl aminopeptidase was detected in lamina propria cells adjacent to the epithelial basal cell layer in man and pig and at the apices of mucous cells in pig submucous glands. Weak glutamyl aminopeptidase activity was confined to capillaries in both species. The findings of this study, along with the ready availability of pig oesophagus, suggest that the pig may be a suitable model for studies of the gullet in man.

Sequence of the large double-stranded RNA segment of the N1 strain of infectious pancreatic necrosis virus: a comparison with other Birnaviridae.

The cDNA sequence of the large dsRNA segment (segment A) of the N1 strain of infectious pancreatic necrosis virus (IPNV) has been determined. The nucleotide and deduced amino acid sequences were compared to the sequences of segment A of the Jasper strain of IPNV and to the sequences of segments A and B (5' and 3' flanking regions) of the 002-73 strain of infectious bursal disease virus (IBDV). The comparison demonstrated that the precursor protein of the major structural polypeptide, pVP2, is highly conserved at the N and C termini, whereas the amino acid sequence of an internal segment shows greater diversity between the strains. This internal segment probably carries the serotype-specific epitopes of birnaviruses. An alternative open reading frame (ORF) (444 bp) partly overlapping with the large ORF (2916 bp) of segment A was found to be conserved among the IPNV strains and is probably also present in the 002-73 strain of IBDV. This small ORF may encode a novel birnavirus polypeptide with an Mr of 17K. SDS-PAGE of radiolabelled purified IPNV particles revealed a band corresponding to the possible novel 17K polypeptide. Short terminal inverted repeats are found in segment A of the N1 and Jasper strains of IPNV and in segment B of the 002-73 strain of IBDV. Segment A of IPNV and segment B of IBDV also contain adjacent inverted repeats at their 5'-terminal flanking regions.

Antibody-mediated enhancement of BK virus infection in human monocytes and a human macrophage-like cell line.

We have monitored BK virus (BKV) antigen expression and multiplication in human monocytes and in a human macrophage (M luminal diameter)-like cell line (U937) in the presence or absence of dilution series of human or rabbit anti-BKV antisera. After infection with BKV alone, restricted expression (structural antigens and T-antigen) and multiplication was recorded in monocytes from some donors, while in U937 cells and monocytes from other donors, no signs of viral activity were detected. Monocyte cultures established from the same donor at different times demonstrated antigen expression/multiplication on two occasions but not on the third. A pronounced enhancement of BKV expression/multiplication in human monocytes and multiplication in U937 cells was seen with some dilutions of all antisera (human and rabbit) used. The pattern of enhancement and the dilution resulting in maximum viral activity was constant and seemed to be determined by the serum, but the exact level of enhancement for a given serum differed considerably in monocytes from different donors and seemed to be determined by the cells. In the latter respect, monocytes taken from the same donor some weeks apart showed variations at the same level, as did cells from different donors. PMA (phorbol-12-myristate-13-acetate) stimulation of monocytes and U937 cells resulted in stronger antibody enhancement in terms of infectivity, without affecting the number of monocytes showing antigen expression. No expression/multiplication of BKV was detected in the murine M luminal diameter-like cell line P338 DI.

Characterization of BK virus-specific antibodies in human sera by Western immunoblotting: use of a zwitterionic detergent for restoring the antibody-binding capacity of electroblotted proteins.

After initial problems related to denaturation of antigenic epitopes, we developed a Western immunoblotting method for the characterization of antibodies reacting with BK virus (BKV) structural polypeptides. When a zwitterionic detergent, Empigen BB, was added to the running buffer during electroblotting, the antibody-binding capacity of electrophoretically separated BKV polypeptides was partially restored. Antibodies reacting with different BKV antigens were detected and visualized by biotinylated anti-species-specific antibodies, peroxidase-conjugated streptavidine, and diaminobenzidine staining. Human sera containing anti-BKV antibodies reacted with VP1, but a serum containing antinuclear antibodies also reacted with VP4, -5 and -6 (histones). Serum from a rabbit inoculated with purified BKV reacted with VP1, and also with VP4, indicating that BKV inoculation may imply production of antibodies against histones.

Characterization of a new serotype of infectious pancreatic necrosis virus isolated from Atlantic salmon.

Virus particles isolated from hatchery reared fish with infectious pancreatic necrosis (IPN) were neutralized by homologous immune sera but not by immune sera raised against IPN virus serotype 1, 2, and 3. This virus isolate, called the N1 strain, was detected in one year old Atlantic salmon (Salmo salar) during an outbreak with histopathological lesions of IPN and slightly increased mortality. The polypeptide pattern of N1 virus differed markedly from that of the three classical IPN virus serotypes. Double stranded RNA isolated from the N1 virus particles, co-migrated during agarose gel electrophoresis with nucleic acid isolated from the IPN virus Jasper and Ab strains. Nucleic acid hybridizations using low stringency washing conditions and a synthetic DNA oligonucleotide probe (representing the 3' end of the A segment of the Jasper strain) gave positive results with the IPN virus Jasper, Ab, Sp, and N1 strains. The results presented in this paper show that the N1 isolate differs immunologically and biochemically from the IPN virus serotypes 1, 2, and 3 and may represent a new serotype of IPNV.

Neutralization test for BK virus: plaque reduction detected by immunoperoxidase staining.

We developed an immunoperoxidase staining test to detect structural antigens of BK virus (BKV) in Vero cell cultures. This test was used to examine the neutralizing activity of human and immunized animal sera. It was shown that sera positive for BKV antibodies measured by hemagglutination inhibition test and enzyme-linked immunosorbent assay (ELISA) were able to prevent expression of BKV structural antigens in cell cultures. The correlation between titers in the hemagglutination inhibition test, levels of BKV IgG measured by ELISA, and the titers assayed by the immunoperoxidase neutralization test was high. We suggest that this type of test may be used instead of conventional neutralization tests for other viruses with slowly developing cytopathogenic effects.

Proteases in normal and diseased human skeletal muscle: a preliminary histochemical survey.

Seven proteases assumed to be aminopeptidases A, B and M, dipeptidyl peptidases II and IV, esteroproteinase and gamma-glutamyltransferase were localized histochemically, using semipermeable membrane simultaneous coupling techniques, in unfixed cryostat sections of skeletal muscle removed from one healthy volunteer, six patients with disuse muscle atrophy, and 15 patients with some form of muscle disease. Normal muscle fibres showed weak reactions for aminopeptidases A and M and for the dipeptidyl peptidases, but no reactivity for gamma-glutamyltransferase or esteroproteinase. No change was detected in diseased muscle fibres except that low gamma-glutamyltransferase and esteroproteinase activities appeared in some cases. The activities of the seven enzymes were stronger in the intermyosial connective tissue than in the muscle fibres, but were also unchanged in disease. The strongest reactions were found in some interstitial cells (mast cells and macrophages) and these were much increased in diseased muscle, particularly for dipeptidyl peptidases II and IV. The findings are interpreted in terms of the release of proteases from such cells and their subsequent involvement in the breakdown of myofibrillar proteins in muscle disease.

Characterization of measles virus-specific antibodies in sera from patients with chronic active hepatitis.

Measles virus-specific antibodies in sera from patients with HBsAg-negative chronic active hepatitis and raised antibody titres against measles virus, have been examined by crossed immunoelectrophoresis. The immunoprecipitates were further analysed by SDS-polyacrylamide gel electrophoresis. Five measles virus-specific precipitation lines were demonstrated using measles virus-infected cells solubilized with Triton X-100. The three major precipitation lines were analysed by SDS-PAGE and contained the virus polypeptides: nucleoprotein, NP (MW approximately 60 000); haemoagglutinin, H (MW approximately 80 000) and fusion protein F1 (MW approximately 40 000). Considerably higher amounts of antibodies against these three virus polypeptides were demonstrated in the patient sera than in sera from healthy controls. By SDS-PAGE analysis of radiolabelled immune complexes adsorbed to Sepharose-protein A, antibodies against five measles virus polypeptides: NP, H, F1, P protein (MW approximately 70 000) and matrix protein, M (MW approximately 37 000) were demonstrated in the patient sera.