Crystal structure of a diabody, a bivalent antibody fragment.

BACKGROUND: Diabodies are dimeric antibody fragments. In each polypeptide, a heavy-chain variable domain (VH) is linked to a light-chain variable domain (VL) but unlike single-chain Fv fragments, each antigen-binding site is formed by pairing of one VH and one VL domain from the two different polypeptides. Diabodies thus have two antigen-binding sites, and can be bispecific. Direct structural evidence is lacking for the connections and dimeric interactions between the two polypeptides of the diabody. RESULTS: The 2.6 A resolution structure has been determined for a bivalent diabody with a flexible five-residue polypeptide linker between the (amino-terminal) VH and (carboxy-terminal) VL domains. The asymmetric unit of the crystal consists of four polypeptides comprising two diabodies; for one of these polypeptides the linker can be traced between the VH and VL domains. Within each diabody the two associated VH and VL domains make back-to-back interactions through the VH domains, and there is an extensive VL-VL interface between the two diabodies in the asymmetric unit. CONCLUSIONS: The structure of the diabody is very similar to that which had been predicted by molecular modelling. Diabodies directed against cell-surface antigens should be capable of bringing together two cells, such as in cell-targeted therapy, because the two antigen-binding sites of the diabody are at opposite ends of the molecule and separated by approximately 65 A.

The 1.9 A crystal structure of a nucleoside diphosphate kinase complex with adenosine 3',5'-cyclic monophosphate: evidence for competitive inhibition.

The X-ray structure of Myxococcus xanthus nucleoside diphosphate (NDP) kinase complexed with adenosine 3',5'-cyclic monophosphate (cAMP) has been determined. The structure was solved by difference Fourier analysis. The refined structure has a crystallographic R-factor of 0.17 at 1.9 A resolution. The phosphoryl group and ribose moiety make extensive polar interactions with the protein, whereas the base interacts only with two hydrophobic residues. The comparison with the structure of the enzyme complex with the substrate adenosine diphosphate (ADP) reported earlier shows that cAMP and ADP interact similarly with the enzyme. The base of the cAMP is present in two conformations, syn and anti, with respect to the sugar. The syn conformer is dominant. Based on the effect of cAMP on phosphorylation of the human NDP kinase NM23, it had been proposed that cAMP might interact with NDP kinase in a manner distinct from other nucleotides. However, the structure of the M. xanthus NDP kinase/cAMP complex indicates that the nucleotide is a competitive inhibitor of the enzyme and occupies the usual nucleotide site. Kinetic assays of the NDP kinase activity in the presence of cAMP were done. Their results are consistent with a competitive character of the cAMP inhibition.

The crystal structure of a human nucleoside diphosphate kinase, NM23-H2.

The 2.8 A resolution X-ray structure of NM23-H2 has been determined by molecular replacement using the structure of Myxococcus xanthus nucleoside diphosphate (NDP) kinase. NM23-H2 is a human NDP kinase. The enzyme catalyses phosphoryl transfer, binds DNA, and can activate the transcription of the c-myc oncogene in vitro. NM23 has also been reported to be a suppressor of metastasis in some types of tumours. Whereas the M. xanthus NDP kinase is a tetramer, NM23-H2 is a hexamer. The fold of NM23-H2 is identical to the fold of other NDP kinases. Two antiparallel helices joined by a turn form one edge of the nucleotide binding cleft. This region moves in a hinge-like fashion in response to substrate binding and crystal packing forces. Additional differences in conformation among the NDP kinases are principally in regions involved in protein-protein contacts within the oligomers. The only protein-protein interaction conserved among all NDP kinases is a dimeric interaction. Several mutations of NM23-H2 have been detected in tumour tissues. These mutations do not involve residues interacting with the substrates, and probably destabilise the enzyme without directly affecting the catalytic activity. Low level phosphorylation of serines has been reported for NM23 both in vitro and in vivo. The structure of the hexamer indicates that two serine residues that have been reported as being phosphorylated, Ser44 and Ser122, are on the surface of the hexamer, and are likely to be phosphorylated by exogenous kinases. In contrast, Ser120 is buried, and is most likely phosphorylated by a direct transfer from the phosphohistidine intermediate of the reaction mechanism.

Nosocomial outbreak of viral hemorrhagic fever caused by Crimean Hemorrhagic fever-Congo virus in Pakistan, January 1976.

This paper describes the clinical, epidemiological, and laboratory investigations undertaken to isolate and identify the etiological agent of a nosocomial cluster of hemorrhagic fever cases due to Crimean hemorrhagic fever (CHR)-Congo virus. Since this virus is usually transmitted by ticks it was surprising that the index case, in a nomadic shepherd, occurred during the winter season when ticks are relatively inactive. These are the first cases of CHF-Congo virus found in humans in Pakistan. Investigations on other biological properties, particularly strain differences and virulence, are being continued at the Islamabad laboratory.

Case-control study of Mastomys natalensis and humans in Lassa virus-infected households in Sierra Leone.

Lassa virus infection and antibodies were studied in households where Lassa fever cases occurred, and compared to those in nearest neighbor houses and "far" houses located across the village from case houses. Seventy-nine percent of all rodents caught in the houses were Mastomys, the natural reservoir of Lassa virus. Rodent infection was not randomly distributed, but rather focal. Thirty-nine percent of the Mastomys in case houses were viremic, compared to 3.7% in control houses. Human antibody prevalence in case houses was 30%, compared to 20% in non-case houses (P less than 0.05, chi-square test, df = 2). Neither seroconversions nor antibody prevalence rates were associated with household size or number of persons per room. Trapping of rodents in half of the case and control houses resulted in a Mastomys reduction ranging from 2.2- to 3.3-fold. This reduction failed to significantly reduce the seroconversion rate to Lassa virus in the people of trapped houses compared to those in untrapped ones. More complete trapping will be needed in order to better evaluate this procedure as a means of interruption of Lassa virus transmission in endemic villages.

Epizootic vesicular stomatitis in Colorado, 1982: infection in occupational risk groups.

In 1982-1983, an epizootic of vesicular stomatitis occurred in the western United States. Veterinarians, research workers, and regulatory personnel who were exposed to vesicular stomatitis virus were examined for patterns of human infection and prevalence of vesicular stomatitis New Jersey serotype neutralizing antibody. Insight into the mechanism of transmission was sought by comparing activities of antibody-positive and antibody-negative persons. A statistically significant risk factor was a history of infected animals sneezing in the face of serosurvey participants. Elevated odds ratios were also calculated for those who usually examined the oral cavity of affected animals, had open wounds on hands or arms, and had exposure to saliva through the eye or skin. Relatively intimate direct contact was required; a higher risk was associated with examining horses than cattle. Neutralizing antibody prevalence was significantly higher among exposed persons with illness (23%) than in exposed persons without a history of clinical illness (7%). Overall, however, infectivity of VSNJ for humans during the epizootic was low.

Epizootic vesicular stomatitis in Colorado, 1982: epidemiologic studies along the northern Colorado front range.

Epidemiologic evaluations were made of farm personnel on vesicular stomatitis-affected premises along the front range of the Rocky Mountains in Colorado during the 1982 epizootic. A similar antibody prevalence was noted to that of veterinarians and research and regulatory personnel who were involved with the same epizootic. Risk of infection resulted from intimate physical contact with infected horses or cows. Incidence and infection rates in horses were 45%; rates in cows were much lower, only 5%. Some epidemiologic clues were gained by a detailed study of an equine ranch. The pasture was incriminated as the area of highest risk, where 100% infection rates were noted. Horses in open pens and barns were at lower risk. Severe clinical disease in horses resulted in higher neutralizing antibody titers than inapparent or mild infection. Maternal antibody was detected in foals up to 4 months of age, and the level of antibody in the foal was a reflection of the dam's antibody level.

Lassa virus hepatitis: a study of fatal Lassa fever in humans.

In order to explore the significance of a previous observation that the most important pathologic changes in fatal Lassa fever are hepatic, we have studied postmortem liver biopsies from 19 patients with fatal Lassa fever. We observed a vigorous macrophage response to cellular damage, but we found no evidence of lymphocyte infiltration in infected hepatic tissues. Using semi-quantitative estimates of liver cell damage, we found a wide range in the severity and progression of Lassa virus hepatitis in our fatal cases. We have classified for descriptive purposes three general nosopoeitic phases: active hepatocellular injury (less than 20% necrosis), continued damage and early recovery, and mitotic activity representing hepatic recovery. We conclude that the liver goes through cellular injury, necrosis and regeneration and any or all may be present at death. In no instance was the degree of hepatic damage sufficient to implicate hepatic failure, and all three phases were represented among our cases. We conclude that the hepatitis of Lassa fever in humans is not the primary cause of death.

Epizootic vesicular stomatitis in Colorado, 1982: epidemiologic and entomologic studies.

An epizootic of vesicular stomatitis (VS) caused by the New Jersey serotype of VS virus affected livestock and humans in 14 western states in 1982-1983. Epidemiological observations were made on at least 10% of the cattle in 4 dairy herds that were located in the vicinity of Grand Junction, Colorado. High rates of neutralizing antibody to the New Jersey serotype were seen in all cattle regardless of whether livestock in the dairy had clinical VS or a decrease in mild production. Antibody titers remained high in these cattle for as long as 2 years after the epizootic. No virus isolations were made from 32 humans with clinical signs compatible with viral disease. Entomological information was obtained during the epizootic from 23 premises in northwestern Colorado. Insect collections yielded 4 isolates from Culicoides spp. midges, 2 from C. variipennis, and 1 each from C. stellifer and C. (Selfia) spp. This is the first report of VS virus isolations from field-collected Culicoides.

Lassa fever in children in Sierra Leone, West Africa.

A systematic study of Lassa fever in febrile children was undertaken over a four-year period, from August 1977 to August 1981, in the Eastern Province of Sierra Leone, West Africa. 479 patients were studied; of these, 245 had adequate specimens to confirm or exclude the laboratory diagnosis of Lassa fever. 51 cases of Lassa fever were identified (21% of patients from whom diagnosis was possible). Virus was isolated from 23 patients. All children had fever; cough and vomiting were present in over 60% of cases studied. The five to nine-year age group had the highest prevalence rate (41% of cases). Seasonal clustering occurred in April, May, and August. A significantly higher proportion of females than males had Lassa fever, a finding which remains to be explained. It is concluded that Lassa fever is a disease of significant concern in the paediatric age group.

A novel 5-lipoxygenase inhibitor prevents gallstone formation in a lithogenic prairie dog model.

Gallstone formation is dependent on biliary cholesterol supersaturation, the pronucleating effects of gallbladder mucin, and inflammation. We evaluated the effect of aspirin (ASA) and a 5-Lipoxygenase inhibitor (FLAPI) on cholesterol precipitation and leukotriene levels in an animal model of cholesterol gallstone formation. Male prairie dogs were divided into four dietary groups: normal chow controls, 1.2 per cent cholesterol (XOL), 1.2 per cent cholesterol plus ASA (XOL + ASA, 100 mg/kg/d), and cholesterol plus FLAPI (XOL + FLAPI, 100 mg/kg/12h). At 3 weeks the subjects were anesthetized, cholecystectomy performed, and the common duct cannulated for bile sampling. Cholesterol precipitation, lithogenic indices, and leukotriene content were analyzed. The group XOL + FLAPI did not form cholesterol crystals, whereas the group XOL + ASA did (P < 0.05, Fisher's exact test). All cholesterol-fed groups had significantly increased lithogenic indices when compared to controls. The XOL + FLAPI group showed a significant and paradoxical increase in LTB4 compared to the other groups (P < 0.05, ANOVA, Fisher's PLSD). This study has shown a significant decrease in the rate of cholesterol stone formation through the use of a novel leukotriene inhibitor at high doses, despite a high cholesterol diet.

Evaluation of an enzyme-linked immunosorbent assay for detection of antibodies to vesicular stomatitis virus in cattle in an enzootic region of Mexico.

An ELISA was compared with the plaque-reduction serum neutralization (PRSN) test, for detection of vesicular stomatitis virus (VSV) antibodies in cattle in a vesicular stomatitis enzootic region of Mexico. A total of 325 bovine serum samples were screened for VSV antibodies. The PRSN test was performed, using Vero cells. The ELISA contained gradient-purified VSV Indiana (Lab strain) and VSV New Jersey (Hazelhurst) as the antigens. Regression analysis and weighted kappa statistic were used to estimate measures of agreement between the 2 assays for detection of VSV antibodies. The ELISA method proved useful for serodiagnosis of vesicular stomatitis. The ELISA and PRSN test results were highly correlated for detection of VSV antibodies.

Epizootic vesicular stomatitis in Colorado, 1982: some observations on the possible role of wildlife populations in an enzootic maintenance cycle.

Sera obtained from wild ungulates, carnivores, and rodents in Colorado were tested for neutralizing (N) antibody against vesicular stomatitis, New Jersey serotype (VSNJ), virus to determine their involvement in the 1982 Colorado VSNJ epizootic in domestic animals. Viremic and N antibody responses of two local rodent species to a 1982 Colorado isolate of VSNJ were determined in the laboratory. The rodents produced only weak viremias, but all developed N antibody. N antibody prevalences for VSNJ in sera from wild ungulates was sufficiently high to indicate their involvement during the epizootic. In addition, the demonstration of N antibody in elk (Cervus elaphus) and mule deer (Odocoileus hemionus) prior to the epizootic in cattle and horses suggests that an enzootic cycle may exist in Colorado.

Serum neutralizing antibody titers in dairy cattle administered an inactivated vesicular stomatitis virus vaccine.

Two doses of a formalin-killed, cell culture-derived vesicular stomatitis virus (vsv)-New Jersey serotype vaccine were administered intramuscularly, 30 days apart, to all lactating and nonlactating cows in a 350-cow dairy herd. Serum specimens were obtained serially from 96 cows before vaccination and at 30, 52 and 80 days after vaccination and from 24 of these cows 175 days after vaccination. Serum neutralizing antibody titers to vsv-New Jersey serotype were determined from serum-dilution, plaque-reduction tests. Serum neutralizing antibody titers also were determined during the same period for 67 nonvaccinated heifers in the herd. Peak group geometric mean serum neutralizing antibody titers of 1:530.46 +/- 1.14 (group geometric mean titer log10, 2.725 +/- 0.055) developed 21 days after the second vaccination, but decreased to a low value of 1:65.36 +/- 1.38 (group geometric mean titer log10, 1.815 +/- 0.142) by 175 days after vaccination. The nonvaccinated group had no detectable antibody titer to vsv-New Jersey serotype throughout the study. All serum specimens from the vaccinates and controls were negative for heterologous reactivity to vsv-Indiana serotype.

A review of rapid prototyping (RP) techniques in the medical and biomedical sector.

The evolution of rapid prototyping (RP) technology is briefly discussed, and the application of RP technologies to the medical sector is reviewed. Although the use of RP technology has been slow arriving in the medical arena, the potential of the technique is seen to be widespread. Various uses of the technology within surgical planning, prosthesis development and bioengineering are discussed. Some possible drawbacks are noted in some applications, owing to the poor resolution of CT slice data in comparison with that available on RP machines, but overall, the methods are seen to be beneficial in all areas, with one early report suggesting large improvements in measurement and diagnostic accuracy as a result of using RP models.

Effectiveness of patient education and psychosocial counseling in promoting compliance and control among hypertensive patients.

Compliance with physician recommendations among long-term hypertensive patients can be a chronic and difficult treatment problem. This study evaluated the relative effectiveness of additional patient education and psychosocial counseling in improving patient compliance. At a family practice clinic, 123 low income, rural, black hypertensive patients were pretested on several psychological characteristics and randomly assigned to one of three groups: vigorous, group patient education and family physician appointments; supportive, individualized psychosocial counseling and family physician appointments; or family physician appointments only, which was the baseline medical care. Intervention and follow-up each lasted three months, and the intervention was in addition to the patients' baseline medical care. Compliance was measured by: keeping follow-up appointments; bringing antihypertension medications to each appointment; consuming these medications; and diastolic blood pressure. Analysis of variance of group mean and change scores, t tests, and chisquare analysis indicated that neither additional patient education nor additional psychosocial counseling improved compliance or blood pressure control significantly better than regular family physician visits alone.

Educational initiatives for cancer nursing in Europe.

The European Communities' "Europe against Cancer" programme proposed that Member States should recognize the specialist nature of oncology and recommended a plan of action relating to the training of health workers in cancer (Action 51 Europe Against Cancer Programme 1987-1989). The Advisory Committee on Training in Nursing arranged a review of training for nurses in cancer to be undertaken throughout Europe. One of the proposals formulated from the findings of this report was to produce a core curriculum and subsequent additional curricula in cancer for trained nurses throughout Europe. Following a workshop of European nurse educators and practitioners early in 1989, a core curriculum was produced by the European Oncology Nursing Society (EONS) and was published in September 1989. This paper outlines the content of that core curriculum and presents EONS' subsequent plans to design educational courses in all aspects of cancer care for European nurses. These courses will be supported by written and audiovisual materials, which may also be used for home learning.