Maternal caesarean section infection (MACSI) in Sierra Leone: a case-control study.

Sierra Leone is the country with highest maternal mortality and infections are the underlying cause in 11% of maternal deaths, but the real burden remains unknown. This study aims to determine the incidence and risk factors of surgical site infection (SSI) post-caesarean section (CS) in women admitted to Princess Christian Maternity Hospital (PCMH) in Freetown, Sierra Leone. A prospective case-control (1:3 ratio) study was implemented from 1 May 2018 to 30 April 2019 and 11 women presenting with suspected or confirmed infection post-CS were screened for inclusion as a case. For each case, three patients undergoing CS on the same day and admitted to the same ward, but not presenting with SSI, were selected as controls. The post-CS infection rate was 10.9%. Two hundred and fifty-four clinically confirmed cases were enrolled and matched with 762 control patients. By multivariable analysis, the risk factors for SSI were: being single (odds ratio (OR) 1.48, 95% confidence interval (CI) 1.36-1.66), low education level (OR 1.68, 95% CI 1.55-1.84), previous CS (OR 1.27, 95% CI 1.10-1.52), presenting with premature membranes rupture (OR 1.49, 95% CI 1.18-1.88), a long decision-incision time (OR 2.08, 95% CI 1.74-2.24) and a high missing post-CS antibiotic doses rate (OR 2.52, 95% CI 2.10-2.85).

Funnel plots and choropleth maps in cancer risk communication: a Delphi study.

BACKGROUND: In the last decades, the issues related to health risk communication to stakeholders and citizens involving health care practitioners and local political authorities have been increasingly debated. The study evaluated an alternative strategy to communicate cancer risk to local communities, involving an expert panel of public health operators in comparing two different graphic tools, Funnel Plot and Choropleth map. STUDY DESIGN: A Delphi method process was implemented to achieve a unified consensus on an expert panel of public health operators with regard to weaknesses and strengths of the Funnel Plot and the Choropleth map as tools for cancer risk communication to local communities and other stakeholders. METHODS: Participants were asked to score the efficacy of the two tools using a scale. Six properties were explored through two consecutive consensus rounds. Scales were used to calculate frequencies and the content validity ratio for each domain within the consensus rounds. RESULTS: After the two consecutive rounds, participants expressed their preference in favour of the Choropleth map for its ability to define the spatial location of the risk and to locate any potential cluster, while reaching a consensus with regard to the Funnel Plot properties to identify hot spots, displaying the scope of the phenomenon under investigation, and to show the precision of estimates and communicating the significance of estimates. CONCLUSIONS: The Delphi process allowed us to conclude that Funnel Plot could be used as a complement to the current and commonly used graphical and visual formats to effectively communicate cancer epidemiological data to communities and local authorities, representing a useful tool for empowering the general population.

Effectiveness of an educational intervention on seasonal influenza vaccination campaign adherence among healthcare workers of the Palermo University Hospital, Italy.

INTRODUCTION: Healthcare workers are continuously exposed to the risk of being infected by influenza viruses during their work, thus representing a threat especially for fragile patients. Although the Italian Ministry of Health strongly recommends influenza vaccination for all HCWs, coverage levels in Italy are still far from the expected. Several studies report that one of the preferred strategies to improve vaccination coverage among Healthcare Workers is improving vaccination knowledge through specific multidisciplinary courses. To assess the effectiveness of an educational intervention on influenza vaccination coverage among Healthcare Workers a study was conducted at "Paolo Giaccone" University Hospital of Palermo, in the occasion of the 2016/2017 seasonal influenza vaccination campaign. MATERIAL AND METHODS: Educational interventions on influenza infection and vaccination were organized involving personnel of the hospital units in which patients were more fragile. The Healthcare Workers who volunteered attend the course were considered as the intervention group, while two controls for each case, composed by Healthcare Workers not attending it, were randomly selected from the same unit. For both groups, a questionnaire was used to investigate attitude and behaviors toward influenza vaccination, while vaccination coverage data were obtained throughout the Hospital informational data system. RESULTS: Overall, out of the 125 participants, 38 (30.4%) followed the course (intervention group) and 87 (69.6%), not attending the course, represented the control group; later, only 43 Healthcare Workers out of 125 (34.4%) underwent vaccination during the season considered. In particular, after the educational intervention, 42% of the attending Healthcare Workers got vaccinated, while vaccination prevalence in the control group was 31%. The Healthcare Workers who underwent vaccination reported, before the intervention, a higher risk perception for contracting (transmitting) influenza compared to those not vaccinated (p<0.05), while no significant difference in risk perception of transmitting influenza to their patients was reported between the two groups. DISCUSSION: Despite the training provided, and an improvement in vaccination adherence by the Healthcare Workers involved, coverage obtained was lower than recommended to reduce influenza spread in hospital contexts. In conclusion, our data suggest that specific training alone may play a role in the improvement of influenza vaccination adherence, but it should be integrated by a wider range of public health measures, including mandatory vaccination.

Current state of genomic policies in healthcare among EU member states: results of a survey of chief medical officers.

Background: A need for a governance of genomics in healthcare among European Union (EU) countries arose during an international meeting of experts on public health genomics (PHG). We have conducted a survey on existing national genomic policies in healthcare among Chief Medical Officers (CMOs) of the 28 EU member states, plus Norway. Methods: A questionnaire was sent to CMOs after a meeting on the policy implications of PHG held during the Italian presidency of the Council of EU in 2014. The survey was closed in November 2015. Results: CMOs response rate was 65.5% (19/29). Twelve (63.2%) reported that their countries had a policy for genomics in healthcare in place, and 15 (78.9%) reported that public funding existed. Public research facilities for the development of such policies were documented in 13 (68.4%) countries, and 15 (83.3%) had working groups devoted to policy development. National agencies carrying out Health Technology Assessment of genomic-based technologies were present in nine countries (50%). Sixteen (88.9%) countries reported having agencies dealing with ethical issues related to genomic technologies. About 55% of countries disclosed the lack of information campaigns aimed at citizens, and 44.4% reported they had a legal framework for direct-to-consumer genetic tests. Conclusion: Belgium, France, Italy, Spain and UK documented the presence of a policy on genomics in healthcare. While many caveats are necessary because of the methodology, results suggest a need for a co-ordinated effort to foster development and harmonization of dedicated policies across EU to responsibly integrate genomics policies into existing health systems.

Improvement in vaccination knowledge among health students following an integrated extra curricular intervention, an explorative study in the University of Palermo.

INTRODUCTION: Vaccination coverages threaten to decrease because of false beliefs in their unsafety and inefficacy. Therefore formation of future health-care workers on this topic is fundamental to deal with any doubt and to promote active immunization among general population. METHODS: In order to assess health-care students' knowledge about vaccination before an integrated seminar on this topic, and to evaluate their improvement after the educational intervention, an integrated educational intervention was held by a multidisciplinary team. Before and after the seminar, 118 students of medicine and biology schools at Palermo University were asked to answer 10 multiple-choice questions regarding vaccine history, mechanism of action, side effects, composition, use and nowadays issues (hesitancy). Two more questions investigating possible changes on students' attitudes towards vaccination and the usefulness of the formative intervention, were added at the post-test phase of the survey. RESULTS: Eighty-one out of 118 students (68.6%) answered to both pre- and post-test questions. 97.6% and 81.5% of the participating group also completed the two additional questions about their improvement in knowledge (question 11) and attitudes (question 12) towards vaccinations. The post-test results showed a significant improvement for all questions administered, except for number 3 (about a specific immunological content), with an overall percentage of correct answers increasing from 38.8% to 77.6% (p©< 0.001). CONCLUSIONS: The present explorative study put the basis for future studies, stronger in the methodology, and highlights the importance of educating health-care professions students by integrated extra-curricular intervention to be held early in their degree curricula and in order to improve knowledge and attitudes towards vaccinations and to prepare them to promote vaccines among the general population.

Phylogenetic analysis in the clinical risk management of an outbreak of hepatitis C virus infection among transfused thalassaemia patients in Italy.

BACKGROUND: Occurrence of hepatitis C virus (HCV) infection is reduced by effective risk management procedures, but patient-to-patient transmission continues to be reported in healthcare settings. AIM: To report the use of phylogenetic analysis in the clinical risk management of an HCV outbreak among 128 thalassaemia outpatients followed at a thalassaemia centre of an Italian hospital. METHODS: Epidemiological investigation and root-cause analysis were performed. All patients with acute hepatitis and known chronic infection were tested for HCV RNA, HCV genotyping, and NS3, NS5A, and NS5B HCV genomic region sequencing. To identify transmission clusters, phylogenetic trees were built for each gene employing Bayesian methods. FINDINGS: All patients with acute hepatitis were infected with HCV genotype 1b. Root-cause analysis, including a lookback procedure, excluded blood donors as the source of HCV transmission. The phylogenetic analysis, conducted on seven patients with acute infection and eight patients with chronic infection, highlighted four transmission clusters including at least one patient with chronic and one patient with acute HCV infection. All patients in the same cluster received a blood transfusion during the same day. Two patients with acute hepatitis spontaneously cleared HCV within four weeks and nine patients received ledipasvir plus sofosbuvir for six weeks, all achieving a sustained virological response. CONCLUSION: Combined use of root-cause analysis and molecular epidemiology was effective in ascertaining the origin of the HCV outbreak. Antiviral therapy avoided the chronic progression of the infection and further spread in care units and in the family environment.

Italian young doctors' knowledge, attitudes and practices on antibiotic use and resistance: A national cross-sectional survey.

OBJECTIVES: Antimicrobial resistance (AMR) is one of the major health issues worldwide. Clinicians should play a central role to fight AMR, and medical training is a pivotal issue to combat it; therefore, assessing levels of knowledge, attitudes and practices among young doctors is essential for future antimicrobial stewardship (AMS) programmes. METHODS: A nationwide, cross-sectional, multicentre survey was conducted in Italy. A descriptive analysis of knowledge and attitudes was performed, along with a univariate and multivariate analysis of their determinants. RESULTS: Overall, 1179 young doctors accessed the survey and 1055 (89.5%) completed all sections. Regarding the knowledge section of the questionnaire, almost all participants declared to know the different species of bacteria proposed, however the percentage of participants who correctly responded to clinical quizzes was 23% for the question on vancomycin-resistant enterococci (VRE), 42% on carbapenem-resistant Enterobacteriaceae (CRE), 32% on extended-spectrum ß-lactamase-producing enterobacteria (ESBL) and 27% on methicillin-resistantStaphylococcus aureus (MRSA). Similarly, 81% of participants disagreed in stating that AMR was adequately addressed during their medical training and 71% disagreed that they received the right example from their tutors. Finally, a high rate of agreement with the proposed actions to combat AMR was documented; in particular, the percentage agreement was 76% for respondents who agreed to be part of an active surveillance system or AMS programme. CONCLUSIONS: Tackling AMR should be a priority for politicians and for all health workers. Inclusion of competencies in antibiotic use in all specialty curricula is urgently needed.

Posttranslational modification of a neurofilament protein during axoplasmic transport: implications for regional specialization of CNS axons.

The possibility that proteins are modified during axoplasmic transport in central nervous system axons was examined by analyzing neurofilament proteins (200,000, 140,000, and 70,000 mol wt) along the mouse primary optic pathway (optic nerve and optic tract). The major neurofilament proteins (NFPs) exhibited considerable microheterogeneity. At least three forms of the " 140,000" neurofilament protein differing in molecular weight by SDS PAGE (140,000-145,000 mol wt) were identified. The "140,000" proteins, and their counterparts in purified neurofilament preparations, displayed similar isoelectric points and the same peptide maps. The "140,000" NFPs exhibited regional heterogeneity when consecutive segments of the optic pathway were separately examined on polyacrylamide gels. Two major species (145,000 and 140,000 mol wt) were present along the entire length of the optic pathway. The third protein (143,000 mol wt) was absent proximally but became increasingly prominent in distal segments. After intravitreal injection of [(3)H]proline, newly synthesized radiolabeled proteins in the "140,000" mol wt region entered proximal mouse retinal ganglion cell (RGC) axons as two major species corresponding to the 145,000 and 14,000 mol wt NFPs observed on stained gels. When transported NFPs reached more distal axonal regions (30 d postinjection or longer), a 143,000 mol wt protein appeared that was similar in isoelectric point and peptide map to the 145,000 and 140,000 mol wt species. The results suggest that (a) the composition of CNS neurofilaments, particularly the "140,000" component, is more complex than previously recognized, that (b) retinal ganglion cell axons display regional differentiation with respect to these cytoskeletal proteins, and that (c) structural heterogeneity of "140,000" NFPs arises, at least in part, from posttranslational modification during axoplasmic transport. When excised but intact optic pathways were incubated in vitro at pH 7.4, a 143,000 NFP was rapidly formed by a calcium-dependent enzymatic process active at endogenous calcium levels. Changes in major proteins other than those in the 145,000-140,000 mol wt region were minimal. In optic pathways from mice injected intravitreally with L-[(3)H]proline, tritiated 143,000 mol wt NFP formed rapidly in vitro if radioactively labeled NFPs were present in distal RGC axonal regions (31 d postinjection). By contrast, no 143,000 mol wt NFP was generated if radioactively labeled NFPs were present proximally in RGC axons (6 d postinjection). The enzymatic process that generates 143,000 mol wt NFP in vitro, therefore, appears to have a nonuniform distribution along the RGC axons. The foregoing results and other observations, including the accompanying report (J. Cell Biol., 1982, 94:159-164), imply that CNS axons may be regionally specialized with respect to structure and function.

Posttranslational processing of alpha-tubulin during axoplasmic transport in CNS axons.

Tubulin proteins in mouse retinal ganglion cell (RGC) neurons were analyzed to determine whether they undergo posttranslational processing during axoplasmic transport. Alpha- and beta-tubulin comprised heterogeneous proteins in the primary optic pathway (optic nerve and optic tract) when examined by two-dimensional (2D) PAGE. In addition, however, alpha-tubulin exhibited regional heterogeneity when consecutive 1.1-mm segments of the optic pathway were analyzed separately. In proximal segments, alpha-tubulin consisted of two predominant proteins separable by isoelectric point and several less abundant species. In more distal segments, these predominant proteins decreased progressively and the alpha-tubulin region of the gel was represented by less abundant multiple forms only; beta-tubulin region of the gel was represented by less abundant multiple forms only; beta-tubulin was the same in all segments. After intravitreal injection of [3H]proline to mice, radiolabeled alpha- and beta-tubulin heteroproteins were conveyed together at a rate of 0.1-0.2 mm/d in the slowest phase of axoplasmic transport. At 45 d postinjection, the distribution of radiolabeled heterogeneous forms a alpha- and beta-tubulin in consecutive segments of optic pathway resembled the distribution of unlabeled proteins by 2D PAGE, indicating that regional heterogeneity of tubulin arises during axonal transport. Peptide mapping studies demonstrated that the progressive alteration of alpha-tubulin revealed by PAGE analysis cannot be explained by contamination of the alpha-tubulin region by other proteins on gels. The results are consistent with the posttranslational processing of alpha-tubulin during axoplasmic transport. These observations, along with the accompanying report (J. Cell Biol., 1982, 94:150-158), provide additional evidence that CNS axons may be regionally specialized.

Alzheimer's disease brain: alterations in RNA levels and in a ribonuclease-inhibitor complex.

A macromolecular alteration occurs at the posttranscriptional level in the Alzheimer's disease (AD) brain. Compared with age-matched controls, total cellular RNA and polyadenylated RNA were substantially reduced in the AD cortex with many neuritic plaques and neurofibrillary tangles. RNA changes are associated with a significant increase in alkaline ribonuclease activity due to an abnormality in the ribonuclease-inhibitor complex. The decrease in protein synthesis in the AD brain, previously observed in patients severely affected with AD, and in translation systems in vitro with AD cortical messenger RNA, may be partly related to an enzyme-inhibitor alteration that affects RNA levels and activity. Decreased protein synthesis therefore may contribute to the characteristic decline in certain neurotransmitter enzymes and to the loss of neurons in the AD brain.

Increased vertical axon numbers in cingulate cortex of schizophrenics.

Data generated from an earlier study have suggested a model in which greater numbers of long, vertical, associative axons may occur in the anterior cingulate cortex of schizophrenic patients relative to control subjects. This hypothesis has now been tested using neuron-specific antibodies raised against the 200-kilodalton neurofilament subunit, a component of neuronal cytoskeleton, to immunostain axons of human postmortem cingulate cortex. A manual method for counting axons in the region of layer II and sublamina IIIA has been designed and applied blindly to parallel control and schizophrenic immunoprocessed specimens. The results show that there are 25% more vertical axons in the schizophrenic than in the control specimens. Preferentially higher numbers of both long vertical axons (62%) and axons associated with blood vessels (52%) have also been noted in the schizophrenic specimens. By contrast, the number of large-caliber horizontal axons was the same in the two groups; therefore, the greater number of vertical axons in schizophrenic specimens does not appear to represent a nonspecific effect. When these data are corrected for the effects of several confounding variables using analysis of covariance, the overall pattern of the results persists. These findings suggest the possibility that there might be an increase of associative inputs into the anterior cingulate cortex of schizophrenic patients, although it is not clear at present whether the differences noted, if replicative, may be primarily or perhaps only secondarily related to the disorder.

Preparation of a recombinant cDNA library from poly(A+) RNA of the Alzheimer brain. Identification and characterization of a cDNA copy encoding a glial-specific protein.

Studies were undertaken to assess the extent to which messenger RNA prepared from the postmortem Alzheimer's disease (AD) brain can be used for the successful preparation of a recombinant cDNA library. Initial experiments focused on the glial-specific marker glial fibrillary acidic protein (GFAP) since GFAP expression appeared to be a model for further studies on mRNAs that may continue to be expressed at high levels in the vicinity of lesioned sites in the AD brain. An AD cDNA library, prepared in the lambda gt11 expression vector system contained GFAP-specific recombinants. One of these was sequenced and the insert was shown to exhibit 88% homology with the similar sequence from mouse GFAP. As established by Northern blots, the size of the GFAP mRNA prepared from the routinely acquired postmortem AD cortex, approximately 2.7 kb, was the same as from a neurologically normal control brain. These results agree with earlier studies on GFAP mRNA from fresh mouse brain. The results demonstrate that in the postmortem AD brain, astroglial-specific mRNA remains sufficiently stable for molecular genetic analysis and may serve as a useful model for examining the genetic expression of mRNAs that may be related to the molecular pathogenesis and the etiology of AD.

Development of an anti-A beta monoclonal antibody for in vivo imaging of amyloid angiopathy in Alzheimer's disease.

We evaluated the efficacy of murine monoclonal antibodies (MAbs) targeted to the A beta amyloid of Alzheimer's disease for development of procedures for the in vivo identification of amyloid angiopathy (AA). MAbs to A beta were prepared and screened for effectiveness in visualizing AA and neuritic plaques in postmortem AD brain sections. They were assessed again after enzymatic cleavage to produce Fab fragments and after labeling with technetium-99m (99mTc) using a diamide dimercaptide ligand system. Modified and radiolabeled Fab fragments retained activity and specificity toward amyloid-laden blood vessels and neuritic plaques. A highly specific murine MAb, 10H3, was identified and characterized that fulfills criteria necessary for the development of an in vivo diagnostic imaging agent. Toxicity studies in rats showed the MAb to be safe. Biodistribution studies in mice demonstrated desirable properties for use as an imaging agent. Expansion and adaptation of these strategies may provide the methods and materials for the noninvasive analysis of AA in living patients, and permit assessment of the contribution of AA to the clinical and pathological features of AD.

Evidence for axonal loss in regions occupied by senile plaques in Alzheimer cortex.

The studies described have sought to determine what, if any, relationship exists between axons and the senile plaque, a hallmark histopathological feature of Alzheimer's disease. A double stain was performed on both early and late Alzheimer frontal cortex tissues in order to examine the interaction between axons stained with antibodies against the 200,000 mol. wt neurofilament subunit (NFP-200) of the axon cytoskeleton and Thioflavin-S, a fluorescent dye that stains plaques. Serial photomicrographs of plaques were taken and axon and plaque profiles were three-dimensionally reconstructed. Analysis of computer-processed images revealed that there were fewer axons within plaques than in regions lying one and two plaque distances away. When axons were observed passing through plaques, swelling and disruption of normal morphology was frequently present. Statistical analyses of axon counts within and around placques showed a gradient of axon density, with increased numbers occurring at progressive distances from the placque. Similar patterns were seen for early and late stages of the disease. The results of this study indicate that disruption of the axonal cytoskeleton may occur within the regions occupied by plaques.

Neurofilament protein-triplet immunoreactivity in distinct subpopulations of peptide-containing neurons in the guinea-pig coeliac ganglion.

A battery of polyclonal and monoclonal antibodies raised against the triplet of identified neurofilament protein subunits was used to investigate neurofilament protein immunoreactivity in neurons of the guinea-pig coeliac ganglion. Using optimal conditions of fixation and tissue processing for each antibody we found that only 20% of the postganglionic sympathetic neurons in the guinea-pig coeliac ganglion contain neurofilament protein-triplet immunoreactivity. Double labelling with neurofilament protein-triplet antibodies raised in different species demonstrated that all of these antibodies labelled the same population of neurons. Double labelling using mouse monoclonal antibodies against neurofilament proteins in combination with rabbit polyclonals to neuronal markers showed that neurofilament protein-triplet immunoreactivity is restricted to specific chemically coded subpopulations of noradrenergic neurons. Approximately 52% of neurons in the ganglion contain neuropeptide Y and are presumed vasomotor neurons projecting to blood vessels in the submucosa of the small intestine. Virtually none of the neuropeptide Y-containing neurons were labelled with neurofilament protein-triplet antibodies. Neurons that contain somatostatin (21%) project to the submucous ganglia of the small intestine. Approximately two-thirds of neurons containing somatostatin are immunoreactive for the neurofilament protein-triplet. The other postganglionic neurons in the ganglion (27%) project to the myenteric plexus of the small intestine and do not contain either neuropeptide Y or somatostatin. Approximately a quarter of these neurons were labelled with neurofilament protein-triplet antibodies. These results suggest that the neurofilament protein-triplet may not be an intrinsic component of the cytoskeleton of all neurons. Furthermore the idea of a chemical coding of neurons should be extended to cytoskeletal proteins. The finding that these neurofilament proteins are confined to specific neuronal subpopulations has important implications for the search for a role of the neurofilament protein-triplet in neurons, for the interpretation of classical neurohistological silver impregnation techniques which appear to stain only neurofilament protein-triplet-containing neurons, as well as for neuropathological conditions that may involve these proteins in disease processes.

Evidence for a diffusional model of Alzheimer amyloid A4 (beta-amyloid) deposition during neuritic plaque formation.

A recent study reported that Alzheimer senile plaques immunostained with monoclonal antibodies against the A4 (beta-amyloid) region of the amyloid precursor protein show gradients of density (Majocha R. E., Benes F. M., Reifel R. L., Rodenrys A. M. and Marotta C. A., Proc. natn. Acad. Sci. U.S.A. 85, 6182-6186, 1988). Although more than one explanation was suggested for this observation, the possible involvement of a diffusional process during plaque maturation was considered. In order to examine this hypothesis, specimens from prefrontal cortex, entorhinal area and hippocampal formation were immunoprocessed in a similar fashion and subjected to quantitative microdensitometric analyses of A4 amyloid reaction product. All plaques in the three brain areas examined showed a curvilinear relationship between the area of amyloid reaction product (expressed in pixel counts) and optical density (expressed as each of six grey scale levels). There was an increase in the area of amyloid at progressively lower density levels. When the area of amyloid reaction product at each density level was correlated with the overall size of individual plaques, it was found that there was a striking increase in the correlation coefficients at progressively lower grey scale levels, with r = 0.853 at the lowest level examined. When a second order derivation of these correlations was performed by expressing individual r-values with respect to an optical density index, an asymptotic relationship resulted with the lowest density levels showing an increasingly sharp rise toward unity. These data are consistent overall with a model for plaque maturation that involves diffusion of amyloid protein through the extracellular space from focal regions of high density where synthesis and/or release may occur.

Development of a monoclonal antibody specific for beta/A4 amyloid in Alzheimer's disease brain for application to in vivo imaging of amyloid angiopathy.

We evaluated the efficacy of murine monoclonal antibodies (Mabs) targeted to beta/A4 amyloid for development of procedures for the in vivo identification of amyloid angiopathy (AA) in Alzheimer's disease (AD). Mabs to beta/A4 amyloid were prepared and screened for effectiveness in visualizing AA and senile plaques in postmortem AD brain sections. They were assessed again after enzymatic cleavage to produce Fab fragments and after labeling with 99mTc using a diamide dimercaptide ligand system. Modified and radiolabeled Fab fragments retained activity and specificity towards amyloid-laden blood vessels and senile plaques. A highly specific murine Mab, 10H3, was identified and characterized that fulfills criteria necessary for the development of a diagnostic imaging agent. Expansion and adaptation of these strategies may provide the methods and materials for the noninvasive analysis of AA in living patients, and permit assessment of the contribution of AA to the clinical and pathological features of AD.

Molecular and cellular biology of Alzheimer amyloid.

Alzheimer's Disease (AD), a disorder of unknown etiology, is the most common form of adult-onset dementia and is characterized by severe intellectual deterioration. The definitive diagnosis of AD is made by postmortem examination of the brain, which reveals large quantities of neurofibrillary tangles (NFT) and senile plaques within the parenchyma. The NFT are composed of paired helical filaments associated with several cytoskeletal proteins. The primary protein component of senile plaques is beta/A4 amyloid, a 42-43 amino acid peptide derived from a much larger molecule, the amyloid precursor protein (APP). Vascular beta/A4 amyloidosis is also prevalent in the disease. The mechanism by which beta/A4 amyloid accumulates in the AD brain is unknown. Recent research has demonstrated that the precursor molecule, APP, is a transmembrane protein with a large extracytoplasmic domain, a membrane spanning region that includes the portion that gives rise to beta/A4 amyloid, and a short intracytoplasmic domain. The precursor has multiple forms among which are those that differ by a variable length insert within the extracytoplasmic domain. The insert has sequence homology to the family of Kunitz protease inhibitor proteins. Cellular and animal models have been developed to study the nature of APP processing and the biological and behavioral consequences of beta/A4 amyloidosis. The results of such studies indicate that the normal processing of APP involves enzymatic cleavage of the molecule within the beta/A4 amyloid region, thus preventing the accumulation of beta/A4 in the normal brain. The factors leading to abnormal processing of APP, and consequent beta/A4 amyloid accumulation within the AD brain, have yet to be identified. In cell culture, the biological effects associated with beta/A4 amyloid include neurotrophic and neurotoxic activities, while the peptide has also been shown to have dramatic behavioral effects in animal models.

Early posttranslational modifications of the three neurofilament subunits in mouse retinal ganglion cells: neuronal sites and time course in relation to subunit polymerization and axonal transport.

We have characterized stages in the posttranslational processing of the three neurofilament subunits, High (NF-H), Middle (NF-M), and Low (NF-L), in retinal ganglion cells in vivo during the interval between synthesis in cell bodies within the retina and appearance of these polypeptides in axons at the level of the optic nerve (optic axons). Neurofilament proteins pulse-labeled by injecting mice intravitreally with [35S]methionine or [32P]orthophosphate, were isolated from Triton-soluble and Triton-insoluble fractions of the retina or optic axons by immunoprecipitation or immunoaffinity chromatography. Within 2 h after [35S]methionine injection, the retina contained neurofilament-immunoreactive radiolabeled proteins with apparent molecular weights of 160, 139, and 70 kDa, which co-migrated with subunits of axonal neurofilaments that were dephosphorylated in vitro with alkaline phosphatase. The two larger polypeptides were not labeled with [32P]orthophosphate, indicating that they were relatively unmodified forms of NF-H and NF-M. About 75% of the subunits were Triton-insoluble by 2 h after isotope injection, and this percentage increased to 98% by 6 h. Labeled neurofilament polypeptides appeared in optic axons as early as 2 h after injection. These subunits exhibited apparent molecular weights of 160, 139, and 70 kDa and were Triton-insoluble. The time of appearance of fully modified polypeptide forms differed for each subunit (2 h for NF-L, 6-18 h for NF-M, 18-24 h for NF-H) and was preceded by the transient appearance of intermediate forms. The modified radiolabeled subunits in optic axons 3 days after synthesis were heavily labeled with [32P]orthophosphate and exhibited the same apparent molecular weights as subunits of axonal neurofilaments (70 kDa, 145 and 140 kDa, and 195-210 kDa, respectively). Whole mounts of retina immunostained with monoclonal antibodies against NF-H in different states of phosphorylation demonstrated a transition from non-phosphorylated neurofilaments to predominantly phosphorylated ones within a region of the axon between 200 and 1000 microns downstream from the cell body. These experiments demonstrate that the addition of most phosphate groups to NF-M and NF-H takes place within a proximal region of the axon. The rapid appearance of modified forms of NF-L after synthesis may imply that processing of this subunit occurs at least partly in the cell body. The presence of a substantial pool of Triton-insoluble, unmodified subunits early after synthesis indicates that the heaviest incorporation of phosphate occurs after neurofilament proteins are polymerized.(ABSTRACT TRUNCATED AT 400 WORDS)

Neuroimaging of vessel amyloid in Alzheimer's disease.

Despite extensive recent advances in understanding Alzheimer's disease (AD) we are unable to noninvasively establish a definite diagnosis during life and cannot monitor the cerebral deposition of amyloid beta protein (A beta) in living patients. We evaluated the use of 10H3, a monoclonal antibody Fab targeting A beta protein 1-28 labeled with Tc-99m. Six subjects with probable AD were studied using single-photon emission computed tomography (SPECT) at times from 0-24 hours following injection. Curves of radioactivity in blood demonstrate a half-life of the injected Fab of 2-3 hours. Images show uptake around the head in the scalp or bone marrow in all subjects. There is no evidence of cerebral uptake of the antibody. Scalp biopsies in all six patients demonstrate diffuse staining with 10H3 of the scalp, a pattern indistinguishable from that found in controls. Evidence of amyloid deposition in the scalp in AD is not seen with other anti-A beta antibodies, suggesting that 10H3 is cross-reacting with another protein. Further studies with anti-A beta antibodies will require longer-lived radionuclides to detect cerebral uptake at later times after injection to allow for complete clearance from the blood. Alternately, imaging using labeled A beta itself may provide a means for noninvasive targeting of cerebral amyloid.