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In response to the pandemic warning provided by the highly pathogenic H5N1 influenza virus infections in Hong Kong, there were world-wide attempts to develop vaccines. Three strategies were followed and although each was associated with some success, there were also some problems. Pre-clinical vaccine efficacy results are presented from one such strategy, that of using an apathogenic H5N3 avian strain for vaccine production.
1. Postnatal mortality in isolated congenital diaphragmatic hernia (CDH) is mainly related to the associated pulmonary hypertension (PH) and to right-to-left shunting. 2. Endothelins (ETs) are potent vasoconstrictors and pro-mitogenic peptides. Strong evidences support their participation in CDH and in the etiology of PH via the activation of ET(A) receptors (ET(A)-Rs). 3. Evaluation of the effect of ABT-627, a selective non-peptidic ET(A)-R antagonist, given from -15 to 210 min post-delivery (1 mg kg(-1) bolus +0.01 mg kg(-1) h(-1) infusion, i.v.), was conducted in the lamb model of CDH. 4. Severity of CDH was assessed in comparison to untreated controls (n=5). Untreated CDH lambs (n=7) had a higher mean pulmonary arterial pressure (MPAP; P<0.0001), lower mean blood pressure (MBP; P=0.0004), higher MPAP / MBP ratio (P<0.0001), lower arterial pH (P<0.0001), higher paCO(2) (P<0.0001), lower paO(2) (P<0.0001) and lower post-ductal pulsatile SaO(2) (P<0.0001) than untreated controls. 5. Treated controls (n=7) showed a higher MPAP, lower MBP, higher MPAP/MBP ratio, lower arterial pH, higher paCO(2), lower paO(2), lower post-ductal pulsatile SaO(2) and lower plasmatic ir-ET ratios compared to untreated controls (P<0.0001). 6. Treated CDH lambs (n=8) showed a higher MBP (P<0.0001), lower MPAP / MBP ratio (P<0.0001), higher arterial pH (P<0.0001), lower paCO(2) (P<0.0001), higher paO(2) (P=0.0228), higher post-ductal pulsatile SaO(2) (P=0.0016) and lower plasmatic ir-ET ratios (P=0.0247) when compared to untreated CDH lambs. 7. These observations revealed that, although acute perinatal treatment with a selective non-peptidic ET(A)-R antagonist had some adverse effects in controls, it attenuated the progressive cardiopulmonary deterioration that occurred after birth in CDH lambs.
BACKGROUND/PURPOSE: The pathophysiology of congenital diaphragmatic hernia (CDH) associated with lung hypoplasia and pulmonary hypertension is not understood fully. Endothelins (ETs) are the most potent vasoconstrictors that also act as promitogenic agents. They may play a role during pregnancy in leading to the condition found at birth and ongoing mortality in CDH. Therefore, the authors studied the effect of CGS 26303, a nonselective endothelin-converting enzyme and neutral endopeptidase inhibitor, in the rat model of CDH. METHODS: Pregnant Sprague-Dawley rats were divided into 3 groups: group 1 (n = 4) received CGS 26303 (50 mg/kg, subcutaneously, twice a day), from gestational day 12 until term (21 to 23 days); group 2 (n = 8) received nitrofen (100 mg/kg, orally) at gestational day 11.5; group 3 (n = 8) received both nitrofen and CGS 26303. The survival of the newborn rats was monitored up to 240 minutes. After natural death or euthanasia, they were weighed and microdissected. The degree of hernia was quantified as small, moderate, or severe, and lungs and liver were harvested and weighed. RESULTS: Newborn rats from mothers of group 3 (n = 81) survived 196 +/- 8 minutes compared with 173 +/- 9 minutes of those of group 2 (n = 97). Severe CDH from group 3 (n = 20) had a mean survival time of 66 +/- 13 minutes compared with 26 +/- 4 minutes for those of group 2 (n = 27). Lung index in severe CDH pups of group 3 was increased by 13% compared with those from group 2 (P < .0001), whereas their liver index went down by 8% (P < .05). CONCLUSIONS: These results suggest that CGS 26303 might have a beneficial effect when given during pregnancy in increasing survival at birth and reducing the severity of the pulmonary hypoplasia in newborn rats with nitrofen-induced CDH.
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BACKGROUND/PURPOSE: Modern trends are toward delayed surgical reduction of congenital diaphragmatic hernia. This study was conducted to verify the hypothesis that the "ease" of ventilation found in the authors' postoperative experience in infants with congenital diaphragmatic hernia (CDH) is associated with postsurgical improvement in pulmonary mechanics. METHODS: Very severe CDH was surgically induced in utero at 90 days' gestation in 31 lambs. At birth pulmonary mechanics (PeDS-Lab) was measured in these preterm lambs with (n = 24) and without (n = 7) early surgical reduction, and in eight non-CDH controls over the same period; functional residual capacity (FRC) also was obtained from 14 of the 31 CDH lambs (seven reduced animals and seven unreduced ones). Management excluded aspiration from the thorax and insertion of chest drains. RESULTS: After 30 minutes of life CDH animals with early surgical reduction demonstrated significantly greater improvement in lung volume, pulmonary mechanics, and oxygenation than those without reduction: FRC, 235% versus 19%; compliance, 57% versus 14%; minute ventilation, 71% versus 30%; and PO2, 143% versus -15%. Over the same period, in preterm controls without CDH, only the compliance varied significantly, demonstrating a 32% increase. CONCLUSION: Based on the mechanics of breathing in these lambs, the authors speculate that neonates with CDH could benefit from early surgical repair because of improvement in pulmonary function, provided extra care is taken to prevent pulmonary overdistension.
In recent years microspheres or microparticles produced from biodegradable polymers such as poly(D,L-lactide) (PLA) and poly(D, L-lactide-co-glycolide) (PLGA) containing encapsulated vaccine antigens have been investigated for administration via parenteral, oral, and intranasal routes. These microparticles allow the controlled release of vaccines with an aim to reduce the number of doses for primary immunisation or to develop single dose vaccines. The polymer materials have been widely regarded as being of minimal toxicity. Evaluation of candidate systems in animal studies have shown antibody levels and cell responses similar to or greater than those observed with adjuvants such as alum. However, there are concerns regarding the integrity and immunogenicity of the antigen during the encapsulation process when the antigen is exposed to organic solvents, high shear stresses and the exposure of antigen to low pH which is caused by polymer degradation. An alternative approach would be to adsorb antigens to the surface of biodegradable polymer particles. Polymeric lamellar substrate particles (PLSP), produced by a simple precipitation of PLA, are suitable for this purpose. The adsorption of antigens onto these particles is a simple procedure. It avoids pH changes due to bulk polymer degradation and the use of solvents and therefore will be less damaging to the vaccine. Moreover, such systems will be much easier to scale up for a clinical study and eventual manufacture. The aim of this article is to discuss the preparation and physical characteristics of PLSP, antigen adsorption, in vivo efficacy of PLSP antigen systems and to consider the potential of PLSP as controlled release adjuvants for protein, peptide or viral vaccines.
Two Thoroughbred horses were presented with various clinical signs which included sweating, agitation, muscle twitching and synchronous diaphragmatic flutter. These signs were associated with profound hypocalcaemia. A diagnosis of primary hypoparathyroidism was made on the basis of low serum ionised calcium concentration, hyperphosphataemia and markedly reduced serum immunoreactive parathyroid hormone concentrations in the presence of normal renal function. Treatment with a combination of intravenous calcium and subsequently oral calcium, magnesium and a vitamin D analogue (dihydrotachysterol) for up to 65 days resulted in complete remission of clinical signs. Horse 1 was euthanased 12 months after the initial recognition of signs. Results of necropsy were unremarkable apart from an absence of detectable parathyroid tissue. Horse 2 returned to athletic activities while receiving only maintenance doses of oral calcium carbonate.
The etiology of congenital diaphragmatic hernia (CDH) is not yet known. Studies in the literature from 1941 have reported that nutritional deficiency of vitamin A during pregnancy could lead to CDH, associated or not with other malformations in young rats. More recently, possible correlations between expression patterns of cellular retinoid-binding protein and retinoic-acid receptors and morphologic effects of vitamin A deficiency have been suggested. The purpose of this study was to verify in human newborns the possible link between vitamin A deficiency and CDH previously observed in experimental animals. Blood samples were obtained during the first hours after birth from 11 term CDH newborns and 11 healthy controls matched for gestational age, and also from 7 mothers in each group, for a total of 7 newborn-mother pairs of matched CDH-controls. Plasma retinol was measured by high-performance liquid chromatography and retinol-binding protein (RBP) by nephelometry. In the 11 matched CDH-control newborns, plasma retinol and RBP levels in CDH newborns were 50% less than control values (P< 0.0002 and <0.006, respectively); in contrast, retinol levels in CDH mothers were significantly higher than those of control mothers (P < 0.005). The observation that the plasma concentrations of retinol and RBP are low in infants with CDH relative to controls may be clinically very relevant and may help to elucidate the mechanism of development of this congenital anomaly.
Lamellar particles and microspheres were produced by precipitation from solutions of resorbable, biocompatible, semi-crystalline poly(L-lactide)[PLA] and amorphous poly(DL lactide co-glycolide)[PLG] copolymer, respectively, to investigate their adjuvanticity towards adsorbed influenza virus. Both types of substrate were capable of adsorbing large quantities of virus (> 15% w/w) and retaining virus (> 60% of the initial load) over an 8 week time scale in-vitro. Potent immune responses were obtained in mice after the intra-muscular injection of adsorbed vaccine systems. The response to virus adsorbed on PLA lamellar particles was almost five times that obtained using PLG microspheres and fourteen times that using aqueous vaccine. The lamellar forms of PLA may function as an immunomodulator enhancing phagocytic activity due to their irregular shape and may be useful in improving the immune response to a variety of protein and viral antigens.
To test the hypothesis that perfluorochemical (PFC) instillation may reduce the pulmonary trauma commonly associated with conventional gas ventilation, we studied 12 lambs with normal lungs and 10 with pulmonary hypoplasia secondary to congenital diaphragmatic hernia (CDH). We used mechanical ventilation for up to 3.5 h, with and without tracheal instillation of LiquiVent PFC liquid. At the end of experimentation lungs were fixed for morphometrical analysis of their components and pulmonary trauma was evaluated by measurement of the perivascular compression index (PCI = % perivascular emphysema/% vessels). In normal lungs good gas exchange and respiratory mechanics were obtained with all modes of ventilation, with no statistical difference in the index of pulmonary trauma with or without instillation of LiquiVent (P >0.05). In the hypoplastic lungs, tracheal instillation of PFC liquid after 30 min of conventional gas ventilation significantly improved PaCO2 (from 107+/-8 to 55+/-6 mmHg, P <0.05), pH (from 7.00+/-0.03 to 7.29+/-0.04, P <0.05), compliance (from 0.08+/-0.01 to 0.25+/-0.03 ml/cmH2O . kg, P <0.05), and ventilatory index (from 1,445+/-148 to 794+/-139, P <0.05). Survival was 6/6 animals with PFC ventilation compared to 1/4 with conventional gas ventilation with no more pulmonary trauma (mean PCI 12.6+/-1.8 vs. 11.4+/-4.0%, P >0.05) for a longer mean ventilatory period in the PFC group. We conclude that the PFC liquid technique of ventilation can improve respiratory physiology when conventional gas ventilation alone is proving inefficient. There was no significant difference in pulmonary trauma at morphometrics between gas and partial liquid ventilation.
To test the hypothesis that perfluorochemical (PFC) instillation may reduce the pulmonary trauma commonly associated with conventional gas ventilation, we studied 12 lambs with normal lungs and 10 with pulmonary hypoplasia secondary to congenital diaphragmatic hernia (CDH). We used mechanical ventilation for up to 3.5 h, with and without tracheal instillation of LiquiVent PFC liquid. At the end of experimentation lungs were fixed for morphometrical analysis of their components and pulmonary trauma was evaluated by measurement of the perivascular compression index (PCI = % perivascular emphysema/% vessels). In normal lungs good gas exchange and respiratory mechanics were obtained with all modes of ventilation, with no statistical difference in the index of pulmonary trauma with or without instillation of LiquiVent (P >0.05). In the hypoplastic lungs, tracheal instillation of PFC liquid after 30 min of conventional gas ventilation significantly improved PaCO2 (from 107+/-8 to 55+/-6 mmHg, P <0.05), pH (from 7.00+/-0.03 to 7.29+/-0. 04, P <0.05), compliance (from 0.08+/-0.01 to 0.25+/-0.03 ml/cmH2O . kg, P <0.05), and ventilatory index (from 1,445+/-148 to 794+/-139, P <0.05). Survival was 6/6 animals with PFC ventilation compared to 1/4 with conventional gas ventilation with no more pulmonary trauma (mean PCI 12.6+/-1.8 vs. 11.4+/-4.0%, P >0.05) for a longer mean ventilatory period in the PFC group. We conclude that the PFC liquid technique of ventilation can improve respiratory physiology when conventional gas ventilation alone is proving inefficient. There was no significant difference in pulmonary trauma at morphometrics between gas and partial liquid ventilation.
OBJECTIVE: To evaluate the rate of absenteeism from work in patients who had undergone open or arthroscopic acromioplasty. DESIGN: A retrospective case series. SETTING: A university hospital. PATIENTS: Eighteen patients with excellent results after open acromioplasty performed by one orthopedic surgeon and 20 patients with excellent results after arthroscopic acromioplasty performed by another orthopedic surgeon. MAIN OUTCOME MEASURE: The time between operation and return to work. RESULTS: There were no statistical differences between the two techniques with respect to the return to work, age, sex and type of work. The overall time off work averaged 203 days (range from 42 to 840 days) for the arthroscopic group compared with 144 days (range from 60 to 540 days) for the open group. CONCLUSIONS: Open acromioplasty, a safe and reliable procedure for the general orthopedic surgeon, is associated with a shorter, thought not significant, delay in return to work than the arthroscopic technique.
Tracheal instillation of perfluorochemical liquid (PFC) lowers surface tension in the lung and thus might reduce barotrauma commonly associated with conventional gas ventilation (GV) in highly immature and hypoplastic lungs. It could be a promising alternative treatment for congenital diaphragmatic hernia (CDH) when GV alone is proving inefficient. The authors compared data for eight newborn lambs with surgically induced CDH. The animals had GV and were studied (in 2 groups) for up to 3.5 hours. Group 1 (GV, n = 4) had gas ventilation only. In group 2 (PFC, n = 4), after 30 minutes of GV, 10 to 12 mL/kg of warmed, oxygenated PFC liquid (LiquiVent) was instilled into the lung via the trachea under pressure-volume curve monitoring. Arterial pressure, blood chemistry, and pulmonary mechanics were evaluated serially; histological analysis was performed. One preassigned animal in group 1 died after 15 minutes. After 30 minutes of life, the cardiopulmonary profile of survivors was indicative of severe respiratory distress (Pao2 < 72 mm Hg with FIO2 at 1.0, PaCO2 > 90 mm Hg, compliance < 0.10 mL/cm H2O/kg) and not different between groups; the severity of pulmonary hypoplasia was further confirmed postmortem; the ratio of lung weight to body weight was 41% of that observed in control lambs, in both gas-only and combined gas/PFC-ventilated animals, compared with their respective controls. After instillation of PFC, there were dramatic improvements in acid-base status and pulmonary compliance in group 2. Survival at 3.5 hours also was markedly different (4 of 4 PFC animals and 1 of 3 GV animals).(ABSTRACT TRUNCATED AT 250 WORDS)
Pre- and post-vaccination sera from 19 volunteers were analysed by the haemagglutination inhibition (HI) test, virus neutralization (VN) assay and avidity enzyme-linked immunosorbent assay (ELISA). The sera were tested against the three strains in a commercial inactivated influenza vaccine; A/Beijing/353/89(H3N2); A/Taiwan/1/86 (H1N1) and B/Yamagata/16/88. Additionally, a range of earlier strains and one newer isolate were assayed for HI- and VN-antibodies. Large variations in the pre-vaccination HI titres were observed for the viruses tested. However, 8-9 days after vaccination HI titres increased to above the assumed protective level (HI > or = 40) in most subjects. Although a limited number of patients were analysed at each sampling point, the time-profile we observed in this study is consistent with data we have obtained in earlier trials (Cox, R.J. et al., Vaccine 1994, 12,993-999). The VN titres, on the other hand, were low against all influenza strains before and up to 6 days, but increased rapidly 8-9 days after vaccination. A recent H3N2 isolate, A/Beijing/32/92 (H3N2), which had drifted further away from the vaccine strain, reacted to low titres or were negative in both the HI and VN assays. No change in the serum avidity to the influenza surface antigens was detected after vaccination, whereas sera from subjects naturally infected with influenza showed an increase in avidity to the infecting virus strain. The increase in serum avidity observed in the infected subjects is probably due to the increased and prolonged antigenic stimulus provided by the replicating virus.
Previous studies have shown that the polysialic acid (PSA) moiety associated with the neural cell adhesion molecule (NCAM) has strong antiadhesive properties that can influence a variety of cell-cell interactions. Based on the size and structure of this long linear homopolymer, we have proposed that the activity of PSA results from its physical properties. In the present study, the premise that the charge and hydration properties of PSA underlie its effects on membrane vesicle aggregation is investigated through the manipulation of ionic strength. The results establish that the antiadhesive properties of PSA are accentuated at low ionic strength, where charge and hydration cause expansion of the polymer size, and are absent at high ionic strengths, where the polymer size collapses. These large effects of PSA on aggregation were shown not to result from changes in the osmolarity of the solvent or from the relatively small effect of ionic strength on the intrinsic functional properties of the adhesion receptors. In addition to this influence on overall membrane-membrane interaction, PSA was found to have a highly localized and ionic strength-sensitive effect on the binding of monoclonal antibodies to NCAM. Together these results suggest that the charge and hydration properties of PSA can impede both molecular interactions between apposing membranes and more direct contact of NCAM with other proteins at the cell surface.
Influenza viruses grown in chicken eggs may comprise mixtures of variants, creating problems in establishing international reference strains and in preparing high growth reassortants. We therefore analyzed representative reference strains of H3N2 viruses from 1987 to 1989 by direct sequencing of HA1. Three of seven reference strains had different nucleotides at the same position in nucleotide gels, indicating the presence of codominant mixtures. These nucleotide duplications occurred at residues previously shown to code for amino acids associated with egg adaptation (156, 186, and 193 of HA1). Cloning of these viruses in chicken eggs permitted separation of the mixtures, and the majority of these cloned viruses could be distinguished with monoclonal antibodies. The remaining four reference strains were homogeneous and contained one of the two amino acids usually found at these residues in HA1 (e.g., 145 Glu or Lys, 186 Ser or Ile). Analysis of epidemic H3N2 isolates, for which mammalian cell and egg isolates are available for sequence analysis from the same patient, confirmed that multiple nucleotide changes can occur at residues associated with egg adaptation. When reference or epidemic strains containing codominant mixtures were passaged in eggs one to five times, one of the codominant strains usually became dominant. Similar results were obtained with Madin Darby Canine kidney (MDCK) cells, although the dominant virus usually differed from that in eggs. Attempts to reselect an influenza virus possessing sequence changes in HA1 characteristic of mammalian cells or the original human isolate (i.e., 156 Glu, 158 Glu, 186 Ser) by multiple passages in MDCK cells were not successful, but evidence was obtained that MDCK cells can provide a selective growth advantage. Thus, variants that are dominant in eggs are not necessarily dominant in MDCK cells. To preserve the original genotype of viruses used as reference strains, we recommend the following procedure: (i) cloning in chicken eggs of the candidate virus at a very early passage, (ii) selection and analysis of multiple clones with the same ferret polyclonal and/or monoclonal antibodies used in the initial screening, and (iii) selection of the isolate whose hemagglutinin molecule most closely resembles the clinical isolate.
Growth of clinical specimens of influenza viruses in eggs can result in the selection of antigenic variants distinct from corresponding viruses grown in mammalian tissue culture. To evaluate the contribution of host cell selection on the antigenic diversity of human influenza isolates, as seen in annual surveillance studies, viruses grown in embryonated eggs were compared by antigenic and genetic analyses with their mammalian tissue culture-grown counterparts. Clinical specimens were gathered from around the world from late 1987 to 1990 and the antigenicity of isolated viruses was assessed by hemagglutination-inhibition assays using immune ferret sera as is currently performed for routine surveillance and the selection of vaccine strains. In addition, viruses were assessed using a panel of anti-H3 HA monoclonal antibodies. The extent of antigenic variation exhibited by the egg-grown strains was far greater than the relative antigenic homogeneity of the tissue culture-grown viruses. Nucleotide sequence analysis of HA1 gene PCR products of 28 MDCK cell and egg derived pairs allowed identification of amino acid substitutions responsible for the antigenic differences observed and the adaptation to growth in eggs. Among these substitutions was a change at amino acid position 186 of HA1 (Ser in tissue culture viruses and lle in egg-grown viruses) which was observed at relatively high frequency. Egg- and MDCK-grown pairs with this single amino acid difference were classified into distinct antigenic groups by ferret sera raised to WHO reference viruses. Given the additional antigenic diversity observed among egg-grown strains, considerable care should be taken in the selection of reference and vaccine strains grown in eggs. Rapid sequence comparisons of MDCK- and egg-grown viruses allow identification of variants arising through egg selection and will prove to be a useful adjunct to antigenic surveillance for the selection of reference and vaccine strains.
Since we stopped inserting ipsilateral underwater chest drains after surgical reduction of the herniated contents in congenital diaphragmatic hernia, both lungs are allowed to expand at their own pace, making it possible to estimate their degree of hypoplasia on postoperative chest x-rays. Thirty-nine consecutive series of postoperative chest x-rays were examined by an independent reviewer in a blind manner and classified into three groups: none to mild pulmonary hypoplasia (group I, 10 cases); moderate to severe (group II, 20 cases); and very severe hypoplasia (group III, 9 cases). Correlations were done among groups and available anatomical and clinical data. Eight patients out of 9 survived in spite of very severe hypoplasia on postoperative chest x-rays, suggesting that indexes of severity may be misleading when they mistake pulmonary hypoplasia for the cause of death.