2α-Methyl-19-nor-(20S)-1,25-dihydroxyvitamin D(3) protects the insulin 2 knockout non-obese diabetic mouse from developing type 1 diabetes without hypercalcaemia.

Type 1 diabetes (T1D) is an autoimmune disease that destroys the insulin-producing beta-islet cells of the pancreas. Currently, there are no treatment modalities for prevention of T1D, and the mechanisms influencing disease inception and early progression are not well understood. We have used the insulin 2(-/-) non-obese diabetic (Ins2(-/-) NOD) model to study stages of T1D and to examine the protective effects of a potent analogue of 1α,25-dihydroxyvitamin D(3), 2α-methyl-19-nor-(20S)-1α,25-dihydroxyvitamin D(3) (2AMD). Pancreatic tissues from control and 2AMD-treated Ins2(-/-) NOD mice were obtained weekly from 5 to 16 weeks of age. Using immunohistochemical (IHC) analysis, samples were analysed for changes in beta cell survival, islet structure and T cell invasion. Weekly intraperitoneal glucose tolerance tests (IPGTT) were performed to assess comparative beta cell function in control and treated animals. IHC demonstrated progressive beta cell destruction in control mice. In contrast, 2AMD treatment preserved islet cell architecture, arrested intra-islet T cell invasion and prevented the transition from insulitis to diabetes. IPGTT results revealed progressive impairment of beta cell function with increasing age in control mice, while 2AMD treatment resulted in normal beta function throughout the study. These results demonstrate that the Ins2(-/-) NOD model provides a rapid and effective method for studying T1D and for assessing efficacy of anti-diabetic agents.

Femoral-facial syndrome: Prenatal diagnosis and clinical features. Report of three cases.

The objective of our article is to illustrate the earliest prenatal sonographic diagnosis of femoral-facial syndrome (FFS) and to illustrate the spectrum of clinical manifestations of this condition. We present serial sonographic evaluation with 3D evaluation in two fetuses diagnosed prenatally with FFS and the postnatal findings in three patients (one fetus following pregnancy interruption and two newborns one of whom was diagnosed prenatally) with FFS. The two patients with prenatally diagnosed FFS were found to have femoral shortening and characteristic facial features, one 12 weeks of gestation, and one at 15 weeks of gestation. The sonographic findings in the two prenatally diagnosed patients were confirmed after delivery. We also present a third patient who was diagnosed at delivery in whom the diagnosis was missed at a routine prenatal sonogram at 19 weeks of gestation. The patients reported herein expand the clinical spectrum of FFS. The utility of sonographic evaluation in diagnosis of the facial appearance and of the bony abnormalities in this condition is emphasized.

The efficacy of an avian metapneumovirus vaccine applied simultaneously with infectious bronchitis and Newcastle disease virus vaccines to specific-pathogen-free chickens.

Avian metapneumovirus (aMPV), Newcastle disease virus (NDV), and infectious bronchitis virus (IBV) are important respiratory pathogens of chickens. To achieve early posthatch protection against all three diseases it would be helpful to deliver live aMPV, IBV, and NDV vaccines simultaneously at 1 day of age. However, previous work has indicated that the efficacy of aMPV vaccines may be affected when codelivered with IBV or NDV vaccines. The efficacy of an aMPV vaccine when codelivered to chickens in a trivalent combination with an NDV and an IBV vaccine was examined. The serological antibody response to the aMPV vaccine given with the IBV and NDV vaccine was significantly lower than when the aMPV vaccine was given alone. However, the aMPV vaccine did not affect the serological response to the IBV and NDV vaccines. Irrespective, the efficacy of the aMPV vaccine was not affected based on clinical signs postchallenge. This is the first report showing aMPV, IBV, and NDV vaccines can be codelivered without affecting the efficacy of the aMPV vaccine.

Onset of immunity following in ovo delivery of avian metapneumovirus vaccines.

Avian metapneumovirus (aMPV) is an important cause of disease in chickens and turkeys. As infection can occur early in life and spread of the virus throughout a flock is rapid, an early onset of immunity post-vaccination would be advantageous. We have studied the serological immune response and the onset of protective immunity of an aMPV vaccine delivered to chickens via the in ovo route compared to oculonasal delivery at day old. A 1000-fold lower dose delivered in ovo to chicken specific pathogen free (SPF) embryos, than vaccination at day old, provided a significantly higher antibody response. In the presence of maternally derived antibody (MDA), there was no significant difference in antibody response between the vaccination routes. However, the onset of immunity (OOI) for the vaccine delivered to MDA positive chicken embryos was 5 days post-hatch in comparison to 8 days post-hatch for the same dose of vaccine given at day old indicating that chicks would be protected against disease earlier in the field if vaccinated by the in ovo route. In further experiments the OOI for a turkey vaccine delivered to MDA positive turkey embryos was shown to be 8 days post-hatch.

Treatment of renal amyloidosis with etanercept in tumour necrosis factor receptor-associated periodic syndrome.

OBJECTIVE: To describe the effect of Etanercept treatment in systemic AA amyloidosis in tumour necrosis factor receptor-associated periodic syndrome (TRAPS). METHODS: Etanercept therapy was given to a 27 year old woman, with systemic amyloidosis and nephrotic syndrome, and to her 51 year old father, also affected by TRAPS, who had previously undergone renal transplant for amyloidosis. Serum SAA levels, plasma cytokines, glomerular filtration rate and serum amyloid P scanning were monitored. RESULTS: Etanercept treatment resulted in initial clinical resolution of nephrotic syndrome in the 27 year old female. Both subjects demonstrated improvements in GFR and initial reduction or stabilisation of amyloid deposits on SAP scanning. CONCLUSION: Etanercept may reverse or slow the progression of systemic AA amyloidosis in subjects with C33Y TNFRSF1A mutation. Treatment may however need to be continuous and life-long to prevent progression to end stage disease.

Endothelial monocyte-activating polypeptide-II (EMAP-II): a novel inducer of lymphocyte apoptosis.

The novel, proinflammatory cytokine endothelial monocyte-activating polypeptide-II (EMAP-II) was first found in tumor cell supernatants. EMAP-II is closely related or identical to the p43 auxiliary protein of the multisynthase complex, which is involved in protein synthesis. In vitro, EMAP-II induces procoagulant activity, increased expression of E- and P-selectins and tumor necrosis factor receptor-1, and ultimately, programmed cell death (apoptosis) in cultured endothelial cells. EMAP-II is also chemotactic for monocytes and neutrophils. However, the role of the p43/EMAP-II cytokine form in tumors is not understood. We hypothesized an immune-regulatory role within neoplastic tissues and investigated its effects on lymphocytes. EMAP-II causes a dose-dependent inhibition of proliferation and apoptosis in Jurkat T cells and mitogen-activated peripheral blood mononuclear cells. Coculture with DLD-1 colorectal cancer cells or media conditioned by these cells induces apoptosis in Jurkat cells, which is partially reversed by antibodies against EMAP-II. Our data suggest that EMAP-II constitutes a component of a novel, immunosuppressive pathway in solid tumors, which is not normally expressed outside the cell but in tumors, may be subject to abnormal processing and released from tumor cells.

Hatchability, serology and virus excretion following in ovo vaccination of chickens with an avian metapneumovirus vaccine.

The present investigation describes for the first time the effect of an avian metapneumovirus vaccine administered in ovo to 18-day-old chicken embryos. The application of the vaccine had no adverse effect on the hatchability or the health of the chicks post hatch. The antibody titres achieved were higher than those determined for birds vaccinated at 1 day old. Not only were the mean titres in the in ovo vaccinated groups higher, but many more birds developed a measurable antibody response than birds vaccinated at 1 day old. Variation of the vaccine dose used in ovo had little effect on the serological responses that peaked 21 to 28 days post hatch. Re-isolation of the vaccine virus was much more successful from birds vaccinated in ovo than from birds vaccinated at 1 day old, and detection of the nucleic acid by polymerase chain reaction correlated with the results of live virus isolation.

Avian metapneumovirus excretion in vaccinated and non-vaccinated specified pathogen free laying chickens.

Vaccinated and non-vaccinated specified pathogen-free White Leghorn laying chickens were challenged at peak of lay by the intravenous or oculonasal route with a virulent avian metapneumovirus (aMPV) subtype B chicken strain. Severe clinical signs and a drop in egg production were induced in the non-vaccinated intravenously challenged birds whereas the vaccinates were not affected. Live virus excretion was demonstrated in the faeces and respiratory tract of non-vaccinated hens for up to 7 days post intravenous challenge. After oculonasal challenge, virus excretion could only be demonstrated in the respiratory tract for up to 5 days. No live virus excretion was found in either the faeces or the respiratory tract of vaccinated birds. Concurrent with live virus isolation, the presence of viral RNA was demonstrated by single reverse transcription-polymerase chain reaction (RT-PCR). Nested RT-PCR was more sensitive and viral RNA could be detected in non-vaccinated birds up to 28 days post either intravenous or oculonasal challenge, at which time the experiment was terminated. Viral RNA was detected for up to 12 days in vaccinated birds. This is the first study investigating excretion of aMPV and viral RNA in vaccinated and non-vaccinated laying hens challenged under experimental conditions. The results are of importance with regard to the persistence of aMPV and the appropriate diagnostic detection method in laying birds.

Assessment of Be1 and Be2 cells in systemic lupus erythematosus indicates elevated interleukin-10 producing CD5+ B cells.

Recent studies indicate that normal B cells can be primed to differentiate into two distinct cytokine-secreting effector subsets, Be1 and Be2. The aim of this study was to analyse, for the first time, Be1 and Be2 cells at the single cell level in SLE patients using the recently developed technique of flow cytometry for intracellular cytokines. Peripheral blood mononuclear cells (PBMC) from SLE patients and age- and sex-matched normal controls were cultured for 24 h in the presence or absence of phorbal myristate acetate and ionomycin (PMA/I) or lipopolysaccharide (LPS). The production of type I (IFN-gamma, IL-2) and type 2 (IL-4, IL-5, IL-6, IL-10, IL-13) cytokines by B cells (and IL-10 production by fractionated CD5+ and CD5- B cells) was investigated using an intracellular cytokine staining technique and flow cytometry. In the absence of PMA/I stimulation, the percentage of B cells from SLE patients was significantly lower than those of normal subjects and significantly more SLE B cells spontaneously produced IL-10 than controls. Moreover, CD5+ B cells from SLE patients were enriched for cells with signs of previous in vivo activation and for high levels of IL-10 production. A significant positive correlation was observed between the percentage of IL-10- and IL-6-producing PMA/I-stimulated B cells in SLE patients, but not in controls. There were no significant differences in the production of other cytokines by B cells of SLE patients and normal subjects. In conclusion, a general alteration of type 1 and type 2 cytokine production by B cells is not observed in SLE patients. The role of B cell cytokines in the pathogenesis of SLE appears to be exerted by elevated secretion of in vivo IL-10, which may play an important role in the immune dysregulation observed in SLE patients. Moreover, the cross regulation of IL-10 and IL-6 is disrupted in SLE patients.

A C-H...O=C hydrogen bond? Intramolecular hydrogen bonding in a novel semirubin.

(4Z)-8-(5-Carboxypentyl)-9-butyl-2,3-diethyl-dipyrrin-1-one (1), a new analogue of xanthobilirubic acid, (4Z)-8-(carboxyethyl)-2,7,9-dimethyl-3-ethyl-dipyrrin-1-one, was synthesized in four steps from the known 2,3-diethyl-dipyrrin-1-one. Whereas xanthobilirubic acid (which is a model for one-half of bilirubin, the yellow pigment of jaundice) and its homologues with hexanoic and longer acid chains at C-8 engage only in intermolecular hydrogen bonding, 1 is found to engage in intramolecular hydrogen bonding. In CDCl(3) solution, dipyrrinone 1 adopts an anti-Z conformation, and its hexanoic acid COOH is hydrogen-bonded to the lactam H-N-C=O and to the pyrrole C(7)-H but not to the pyrrole NH. The latter constitutes an example of a hydrogen bond of the type C-H...O=C, weak and detected typically in crystals. Dipyrrinone 1 is found by vapor pressure osmometry to be monomeric in CHCl(3), but its methyl ester (2) tends toward being dimeric, like that of methyl xanthobilirubinate, which is dimeric.

Retroviral activity in Behçet's syndrome and systemic lupus erythematosus detected by a PCR-based reverse transcriptase assay.

The objective of this study was to assess retroviral activity in Behçet's syndrome (BS) and systemic lupus erythematosus (SLE) patients. Serum and peripheral blood mononuclear cells (PBMC) were obtained from patients and normal volunteers and PBMC cultured with and without phytohaemagglutinin stimulation. Reverse transcriptase (RT) activity in serum and culture supernatants was measured using a sensitive polymerase chain reaction-based assay. An RT activity above the levels in normal controls was detected in a minority of patients with BS (2/15) and SLE (1/13) and was typically present in all three types of sample. Elevated levels of RT activity were not detected in follow-up samples from the two BS patients. Our findings indicate that elevated RT activity is present in only a minority of patients with BS and SLE. Simultaneously elevated activity in all three sample types implicates PBMC as the source of this retroviral activity.

Electrical stimulation of transforming growth factor-beta 1 secretion by human dermal fibroblasts and the U937 human monocytic cell line.

The in vitro effects on human dermal fibroblasts and the U937 human monocytic cell line of three phases of electrical microcurrents generated by the ACE Stimulator were investigated. The growth and viability of growing and confluent dermal fibroblasts were not directly influenced by the separate microcurrent phases. One form of microcurrent (designated phase 1) stimulated both dermal fibroblasts and U937 cells to secrete transforming growth factor-beta 1 (TGF-beta 1), which is an important regulator of cell-mediated inflammation and tissue regeneration, but none of the three phases stimulated secretion of the pro-inflammatory cytokine interleukin-6 by U937 cells. The stimulation of TGF-beta 1 secretion in these experiments was not dramatic (a median increase over control levels of 20-30%), although it could be biologically significant.

Quantitative and qualitative analysis of the balance between type 1 and type 2 cytokine-producing CD8(-) and CD8(+) T cells in systemic lupus erythematosus.

The production of type 1 (IFN-gamma, IL-2) and type 2 (IL-4, IL-5, IL-10, IL-13) cytokines by CD8(-) and CD8(+) T cells from systemic lupus erythematosus (SLE) patients and normal subjects was investigated using an intracellular cytokine-staining technique. This flow cytometric method facilitates analysis of both surface markers and cytoplasmic cytokines, after a short term (6 h) culture with or without phorbol myristate acetate and ionomycin (PMA/I) stimulation. In SLE patients, more unstimulated T cells produced IL-10 in comparison with controls; other cytokines were not detected in unstimulated cells. The percentage of IL-10-secreting T cells did not significantly increase after PMA/I stimulation of cells from SLE patients. The mean intensity of fluorescence (MIF) of intracellular IL-4 staining was significantly higher in CD8(-) T cells of SLE patients than controls. Significantly fewer CD8(-) and CD8(+) T cells from SLE patients secreted IFN-gamma after PMA/I stimulation compared with controls. The MIF and percentage of IL-2, IL-5, and IL-13-secreting cell subsets were not significantly different between SLE patients and controls. These findings indicate that T cells of SLE patients are already stimulated to produce IL-10 in vivo, which may result in downregulation of IFN-gamma secreting CD8(-) and CD8(+) T cells observed following PMA/I stimulation. Thus, the population size of Th1 and Tc1 cells are reduced in SLE patients whereas the effector function of Th2 cells, with respect to IL-4 production, is enhanced in SLE patients. Furthermore, although the balance between Th1/Th2 and between Tc1/Tc2 is disrupted in SLE patients, it is significantly biased in favour of the Th2 subset only.

Infectious bronchitis virus vaccine interferes with the replication of avian pneumovirus vaccine in domestic fowl.

Experiments were performed in chickens to ascertain whether application of infectious bronchitis (IB) H120 vaccine had an effect on the replication of an attenuated avian pneumovirus (APV) strain, using as indicators virus detection, humoral antibody responses and clinical protection against in vivo APV challenge. A preliminary experiment demonstrated that pharyngeal swabs were as efficient for recovery of APV as were buccal cavity swabs, and that either site was superior to swabbing the nasal cavity. APV was detected to a similar extent by both a reverse transcriptase-polymerase chain reaction (RT-PCR) and virus isolation; therefore, RT-PCR was used in subsequent experiments. In chickens vaccinated with APV alone, APV was detected by RT-PCR in most birds for 1 week after vaccination. When IB vaccine had been applied 1 week earlier, APV detection was delayed and much reduced. This interference by IBV resulted in a lower APV antibody response to vaccination. Following challenge with virulent APV, birds that had been vaccinated with APV alone were fully protected both clinically and virologically. Chickens that had received both vaccines were still protected clinically, but challenge virus could be detected in some pharyngeal swabs 4 days after challenge. In contrast, the APV vaccine had no effect on either the antibody response to the IB vaccine or the level of protection against IB challenge. It is concluded that IB vaccination interferes with the replication of APV, resulting in a reduction in the antibody response but with no adverse effect on the induction of protective immunity.

Protection of chickens against renal damage caused by a nephropathogenic infectious bronchitis virus.

The ability of the infectious bronchitis (IB) Ma5 and 4/91 live-attenuated vaccines to protect against kidney damage caused by a nephropathogenic strain of IB virus (B1648) was investigated. Protection parameters considered were gross and microscopic renal pathology, and the use of a polymerase chain reaction to detect IB RNA in kidney tissue. By each parameter, Ma5 vaccine alone provided poor protection, but 4/91 alone or the combined program both protected well.

Cloning, expression and immunogenicity of the avian pneumovirus (Colorado isolate) F protein.

The F protein of the Colorado isolate of avian pneumovirus (APV), expressed from a DNA plasmid, was recognized by antiserum to both A and B subgroup APVs. After two intramuscular injections of turkeys with this plasmid, a homologous antibody response was detected by enzyme-linked immunosorbent assay. This antibody also recognized subgroup A APV. However, there was no neutralization of the Colorado isolate or of subgroup A or B viruses. Although no significant clinical protection was detected following homologous challenge of poults, an anamnestic serological response was seen, suggesting that a systemic antibody response but no local mucosal immunity was induced.

Semirubin. A novel dipyrrinone strapped by intramolecular hydrogen bonds.

(4Z)-9-(5-Carboxypentyl)-2,3,7,8-tetramethyl-(10H)-dipyrrin- 1-one (1, semirubin), a new dipyrrinone model for one-half of bilirubin, the yellow-orange neurotoxic pigment of jaundice, was synthesized following Friedel-Crafts acylation of 2,3,7, 8-tetramethyl-(10H)-dipyrrin-1-one (5) with the half-ester acid chloride of adipic acid. Unlike other dipyrrinone models for bilirubin, such as the xanthobilirubic acids, which engage only in intermolecular hydrogen bonding, 1 is unique in having been designed and found to engage in intramolecular hydrogen bonding, between the carboxylic acid and the dipyrrinone lactam and pyrrole. This important conformation-determining structural characteristic, shared by 1 and bilirubin, renders them less polar than their methyl esters and leaves them monomeric in nonpolar solvents, where their esters are dimeric. The corresponding 10-oxo analogue (3) of 1 serves as a model for 10-oxo-bilirubin, a presumed bilirubin metabolite in alternate pathways for bilirubin excretion. Like 1, 3 is found to engage in intramolecular hydrogen bonding. Unlike the methyl ester of 1, the ethyl ester of 3 is not intermolecularly hydrogen bonded in nonpolar solvents.