Super-resolution microscopy as a potential approach to diagnosis of platelet granule disorders.

BACKGROUND: Many platelet functions are dependent on bioactive molecules released from their granules. Deficiencies of these granules in number, shape or content are associated with bleeding. The small size of these granules is such that imaging them for diagnosis has traditionally required electron microscopy. However, recently developed super-resolution microscopes provide sufficient spatial resolution to effectively image platelet granules. When combined with automated image analysis, these methods provide a quantitative, unbiased, rapidly acquired dataset that can readily and reliably reveal differences in platelet granules between individuals. OBJECTIVE: To demonstrate the ability of structured illumination microscopy (SIM) to efficiently differentiate between healthy volunteers and three patients with Hermansky-Pudlak syndrome. METHODS: Blood samples were taken from three patients with Hermansky-Pudlak syndrome and seven controls. Patients 1-3 have gene defects in HPS1, HPS6 and HPS5, respectively; all controls were healthy volunteers. Platelet-rich plasma was isolated from blood and the platelets fixed, stained for CD63 and processed for analysis by immunofluorescence microscopy, using a custom-built SIM microscope. RESULTS: SIM can successfully resolve CD63-positive structures in fixed platelets. A determination of the number of CD63-positive structures per platelet allowed us to conclude that each patient was significantly different from all of the controls with 99% confidence. CONCLUSIONS: A super-resolution imaging approach is effective and rapid in objectively differentiating between patients with a platelet bleeding disorder and healthy volunteers. CD63 is a useful marker for predicting Hermansky-Pudlak syndrome and could be used in the diagnosis of patients suspected of other platelet granule disorders.

T cell phenotypes in patients with common variable immunodeficiency disorders: associations with clinical phenotypes in comparison with other groups with recurrent infections.

Common variable immunodeficiency disorders (CVID) are a group of heterogeneous conditions that have in common primary failure of B cell function, although numerous T cell abnormalities have been described, including reduced proliferative response and reduced regulatory T cells. This study compared the T cell phenotype of CVID patients subdivided into clinical phenotypes as well as patients with partial antibody deficiencies [immunoglobulin (Ig)G subclass deficiency and selective IgA deficiency], X-linked agammaglobulinaemia (XLA) and healthy and disease controls. Absolute numbers of T cell subpopulations were measured by four-colour flow cytometry: naive T cells, central and effector memory and terminally differentiated (TEM) T cells, using CD45RA and CCR7 expression. Early, intermediate and late differentiation status of T cells was measured by CD27/CD28 expression. Putative follicular T cells, recent thymic emigrants and regulatory T cells were also assessed. Significant reduction in naive CD4 T cells, with reduced total CD4 and recent thymic emigrant numbers, was observed in CVID patients, most pronounced in those with autoimmune cytopenias or polyclonal lymphoproliferation. These findings suggest a lack of replenishment by new thymically derived cells. CD8 naive T cells were reduced in CVID patients, most significantly in the autoimmune cytopenia subgroup. There was a reduction in early differentiated CD4 and CD8 T cells and increased CD8 TEM in the CVID patients, particularly autoimmune cytopenia and polyclonal lymphoproliferation subgroups, suggesting a more activated T cell phenotype, due perhaps to an antigen-driven process. XLA patients had significantly reduced putative follicular T cells, which may depend on B cells for survival, while no significant alterations were observed in the T cells of those with IgG subclass deficiency or selective IgA deficiency.

Modelling the within-host growth of viral infections in insects.

Insects are infected by a variety of pathogens, including bacteria, fungi and viruses, which have been studied largely for their potential as biocontrol agents, but are also important in insect conservation (biodiversity) and as model systems for other diseases. Whilst the dynamics of host-pathogen interactions are well-studied at the population level, less attention has been paid to the critical within-host infection stage. Here, the reproductive rate of the pathogen is largely determined by how it exploits the host; the resources supplied by the host in terms of size and condition; competition with other pathogens; and the speed with which it kills the host (death being an inevitable outcome for obligate-killing pathogens). In this paper we aim to build upon recent developments in the literature by conducting single infection bioassays to obtain data on growth and fitness parameters for phenotypically different and similar strains of nucleopolyhedroviruses in the Lepdipoteran host Spodoptera exigua. Using these data, a simple mechanistic mathematical model (a coupled system of differential equations) is derived, fitted and parameter sensitivity predictions are made which support empirical findings. We unexpectedly found that initial growth of virus within the host occurs at a double-exponential rate, which contrasts with empirical findings for vertebrate host-pathogen systems. Moreover, these infection rates differ between strains, which has significant implications for the evolution of virulence and strain coexistence in the field, which are still relative unknowns. Furthermore, our model predicts that, counter to intuition, increased viral doses may lead to a decrease in viral yield, which is supported by other studies. We explain the mechanism for this phenomenon and discuss its implications for insect host-pathogen ecology.

Btn1 affects cytokinesis and cell-wall deposition by independent mechanisms, one of which is linked to dysregulation of vacuole pH.

btn1, the Schizosaccharomyces pombe orthologue of the human Batten-disease gene CLN3, is involved in vacuole pH homeostasis. We show that loss of btn1 also results in a defective cell wall marked by sensitivity to zymolyase, a beta-glucanase. The defect can be rescued by expression of Btn1p or CLN3, and the extent of the defect correlates with disease severity. The vacuole and cell-wall defects are linked by a common pH-dependent mechanism, because they are suppressed by growth in acidic pH and a similar glucan defect is also apparent in the V-type H(+) ATPase (v-ATPase) mutants vma1Delta and vma3Delta. Significantly, Btn1p acts as a multicopy suppressor of the cell-wall and other vacuole-related defects of these v-ATPase-null cells. In addition, Btn1p is required in a second, pH-independent, process that affects sites of polarised growth and of cell-wall deposition, particularly at the septum, causing cytokinesis problems under normal growth conditions and eventual cell lysis at 37 degrees C. Thus, Btn1p impacts two independent processes, which suggests that Batten disease is more than a pH-related lysosome disorder.

Circadian distribution of hematology variables in subjects with multiple sclerosis.

Hematology variables were measured in blood samples obtained every 3h (8/24h) from 10 multiple sclerosis (MS) patients and 34 healthy subjects and analyzed for circadian characteristics using the population multiple-components method. Red blood cell (RBC) and hemoglobin levels as well as hematocrits exhibited circadian rhythms with minimal amplitudes in healthy individuals and insignificant variability in the smaller group of MS patients. In contrast the total white blood cell (WBC) and platelet counts for MS patients and healthy individuals both showed significant circadian characteristics while the mean 24h WBC and platelet levels did not significantly differ between the two groups. When the different WBC subsets were examined independently, statistically significant circadian rhythms were seen for lymphocytes and eosinophils for both MS patients and healthy individuals and for neutrophils only in the latter. Moreover, the 24h mean levels of lymphocytes, basophils, and eosinophils were significantly higher for the healthy controls while those of monocytes were higher for the MS patients. However, of all the variables tested with significant circadian rhythms in both groups of individuals, only those of lymphocyte numbers exhibited different patterns with somewhat higher amplitude in healthy individuals and a peak level occurring over an hour after that of MS patients. These changes may be the reflection of a disturbance in the regulation of patterns of lymphocyte activity and migration in MS patients. In addition, the elevation in circulating monocytes in MS patients is consistent with the inflammatory nature of the disease.

Phenotypic and genotypic characterisation of persistent baculovirus infections in populations of the cabbage moth (Mamestra brassicae) within the British Isles.

The genotypic relatedness of persistent baculovirus infections within UK populations of Mamestra brassicae was assessed by sequencing amplified regions from polyhedrin and ie1. Most populations harboured Mamestra brassicae (Mb) nucleopolyhedrosis virus (NPV) which showed very little genotypic variation between populations. However, one population harboured a virus that closely resembled a baculovirus found previously only in Pine Beauty Moth (Panolis flammea) populations in Scotland. Persistent baculoviruses that had emerged spontaneously as lethal, overt infections from two of the insect populations were compared with the type strain of MbNPV and a mixture of P. flammea (Pafl) NPV strains, isolated from a single host, by bioassay in virus-free Spodoptera exigua larvae. Reactivated baculoviruses were as pathogenic as the stock virus and showed phenotypic characteristics closest to the type strain they most resembled genetically. Sequence data from the insect host cytochrome oxidase genes were compared and showed a high degree of sequence conservation between populations and it was not possible to determine whether the persistent baculovirus infections had arisen on many occasions or whether they represented a single initial infection that had spread with the host. However, the presence of two distinct virus genotypes in separate M. brassicae populations suggests multiple colonisations of the host are a possibility.

Vertical transmission of sublethal granulovirus infection in the Indian meal moth, Plodia interpunctella.

Knowledge of the mechanisms of pathogen persistence in relation to fluctuations in host density is crucial to our understanding of disease dynamics. In the case of insect baculoviruses, which are typically transmitted horizontally via a lifestage that can persist outside the host, a key issue that remains to be elucidated is whether the virus can also be transmitted vertically as a sublethal infection. We show that RNA transcripts for the Plodia interpunctella GV granulin gene are present in a high proportion of P. interpunctella insects that survive virus challenge. Granulin is a late-expressed gene that is only transcribed after viral genome replication, its presence thus strongly indicates that viral genome replication has occurred. Almost all insects surviving the virus challenge tested positive for viral RNA in the larval and pupal stage. However, this proportion declined in the emerging adults. Granulin mRNA was also detected in both the ovaries and testes, which may represent a putative mechanism by which reduced fecundity in sublethally affected hosts might be manifested. RNA transcripts were also detected in 60-80% of second-generation larvae that were derived from mating surviving adults, but there was no difference between the sexes, with both males and females capable of transmitting a sublethal infection to their offspring. The data indicate that low-level persistent infection, with at least limited gene expression, can occur in P. interpunctella following survival of a granulovirus challenge. We believe that this is the first demonstration of a persistent, sublethal infection by a baculovirus to be initiated by a sublethal virus dose. We hypothesize that the 'latent' baculovirus infections frequently referred to in the literature may also be low level persistent, sublethal infections resulting from survival from initial baculovirus exposure.

Use of homozygosity mapping to identify a region on chromosome 1 bearing a defective gene that causes autosomal recessive homozygous hypercholesterolemia in two unrelated families.

Familial hypercholesterolemia (FH) is a common inherited disorder of metabolism characterized clinically by high levels of low-density lipoprotein (LDL) in plasma owing to reduced catabolism. This leads to accelerated atherosclerosis and thus to an increased risk of coronary heart disease. FH is usually caused by defects in the gene for either the LDL receptor or apolipoprotein B (apoB), the ligand for the LDL receptor. Elsewhere, we have described two unrelated patients with phenotypic homozygous FH. Both patients were offspring of consanguineous unions, and linkage to either the gene for the LDL receptor or the gene for apoB was excluded in both. Their cells in culture do not degrade LDL, despite the presence of normal surface binding of LDL to the LDL receptor. This observation suggests that the patients may be homozygous for a defective gene that encodes a component of the internalization pathway. We first excluded linkage of the defect to known genes for proteins reported to be involved in internalization of receptors in clathrin-coated pits. We then performed genomewide homozygosity mapping. Genotyping of 500 polymorphic markers in three affected and seven unaffected members of the first pedigree showed that recessive hypercholesterolemia in this family is localized to a single chromosomal region on 1p36-p35. Genotyping of two affected and five unaffected members of the second pedigree provided further evidence of linkage to this locus, thereby mapping the disease-causing gene to a 12-cM region on chromosome 1p36-p35, with a combined LOD score of 5.3 in these unrelated families. Identification of the gene in this region may lead to new insights into the mechanisms of LDL receptor-mediated uptake of LDL by cells and may help to identify further genetic risk factors for premature atherosclerosis.

Eye injuries in the U.S. Armed Forces.

OBJECTIVE: To develop prevention and treatment modalities for eye injuries, ophthalmologists require epidemiological data on the various types of eye injuries. This study sought to define eye injury patterns in the U.S. armed forces. METHOD: Data on patterns of eye injury in the armed forces were obtained through voluntary reporting by U.S. military ophthalmologists throughout the world. The reporting format was standardized with the U.S. Eye Injury Registry initial and follow-up report forms. The data were analyzed for significant injury patterns. RESULTS: Data on 112 patients were submitted, representing a broad range of the military population. Data on a total of 96 patients with a 6-month follow-up were analyzed in this study. Immediately after injury, 43% of the patients were noted to have poor vision (worse than 20/200). After treatment, only 20% were noted to have poor vision. Patients lost an average of 21.6 days of work after a severe eye injury. CONCLUSION: An eye injury is a traumatic and potentially debilitating event. The loss of visual acuity can be drastic, resulting in an extensive recovery period.

Expectancies, evaluations and attitudes: prediction of college student drinking behavior.

OBJECTIVE: The goal of this study was to investigate a model of college student alcohol use that not only included primary demographic and social factors shown to influence college student drinking behavior but also measured the influence of expectancies, evaluations of expectancies, and attitudes in prospectively predicting drinking behavior. METHOD: Participants (N = 17) were recruited from an introductory psychology course subject pool and were asked to complete several questionnaires, including a modified Alcohol Expectancy Questionnaire, an attitude questionnaire and a modified timeline follow-back measure. These measures were completed at baseline and at a 1-month follow-up assessment. The study was then replicated with a separate sample (N = 162). RESULTS: The results of a series of mixed regression analyses indicated that, after accounting for demographic variables, social norms and a college lifestyle attitude variable, the only significant predictor consistent across drinking measures was the general attitude variable. This was true for both the prospective and concurrent analyses of alcohol use. The evaluations of expectancies did account for a significant portion of the variance but appeared secondary to the general attitude measure. The results of the study were replicated in a separate sample. CONCLUSIONS: Contrary to previous research, the results of this study suggest that attitudes toward alcohol use account for more variance in predicting drinking behavior than both alcohol expectancies and evaluations of those expectancies. The results of this study are also consistent with findings that evaluations of alcohol-related expectancies predict drinking behavior.

Infectivity, speed of kill, and productivity of a baculovirus expressing the itch mite toxin txp-1 in second and fourth instar larvae of Trichoplusia ni.

A cDNA clone of the gene coding for the paralytic neurotoxin (tox34) from the female straw itch mite, Pyemotes tritici, was created by RT-PCR and inserted into the genome of the Autographa californica nucleopolyhedrovirus (AcMNPV) under the control of the AcMNPV p10 promoter. This recombinant virus, AcTOX34.4, caused a rigid paralysis in infected larvae. The infectivity of AcTOX34.4 was compared to the wild-type parent strain, AcMNPV-C6, in second and fourth instar larvae of the cabbage looper, Trichoplusia ni. There were no significant differences in LD(50) values between the recombinant virus and its wild-type parent strain but, as expected, the LD(50) was lower for second instar larvae. The mean time to death and yield of occlusion bodies were measured in second and fourth instar T. ni larvae at a high (100% mortality) and low (<50% mortality) doses of the virus. The mean time to death of recombinant infected larvae was reduced by 50-60% compared to larvae infected with the wild-type strain, depending on virus dose and instar, with these larvae becoming paralysed after approximately 60 h and dying 10-20 h later. This is among the fastest speeds of kill recorded for recombinant baculoviruses. Fourth instar larvae were found to succumb to the recombinant virus more quickly than the second instar larvae. The increase in the speed of kill of the recombinant virus was accompanied by a large reduction of approximately 95% in the yield of progeny virus. The yield of virus showed a highly significant relationship with time to death, but this relationship was complex and varied between the different viruses, concentrations, and instars. The yield per unit weight of the larvae was found to be constant at a low virus dose and increased over time at a high virus dose, irrespective of instar and virus. It is predicted that these changes in the performance of the recombinant virus would act toward reducing its fitness, leading to it being outcompeted by the wild type in field situations.

Propofol and pain on induction: the effect of injectate temperature in children.

A prospective, randomized, double-blind study was conducted to determine whether cooled intravenous propofol reduces the incidence of pain on induction of anaesthesia in children. Seventy patients aged 3-10 years, ASA I or II, were randomized to receive 1% propofol plus 0.05% lidocaine either at room temperature (20-23 degrees C) or cooled to 4C. Pain was assessed by a blinded observer using a behavioural scale. Data from 69 patients were analysed. The incidence of pain was 9/34 (26%, 95% confidence intervals 11-41%) in the room temperature injectate group and 5/35 (14%, 95% confidence intervals 3-26%) in the cold injectate group. These differences were not statistically significant (P=0.21, chi-squared test). Pain scores were similar in both groups.

Synthesis and biological activities of position one and three transposed analogs of the opioid peptide YKFA.

Tyr-c[D-Lys-Phe-Ala], YKFA, is a potent opioid peptide analog with subnanomolar IC50s toward mu and delta receptors. Transposing Phe and Tyr, a modification found to promote mu antagonist activity in opioid/somatostatin hybrids, gave surprisingly high mu agonist activities for several related analogs, considering the lack of a 1-position hydroxyl function.

A case of successful use of reproductive technology for a couple with subfertility due to retrograde ejaculation, where artificial insemination had failed.

A case history of a couple where the infertility was due to retrograde ejaculation is presented; when conventional treatment with artificial insemination using sperm isolated from postejaculatory urine failed, IVF and GIFT technology was applied. This resulted in a pregnancy which unfortunately failed at 3 months, unrelated to the method of conception. This case report highlights the possible use of IVF/GIFT for yet another type of male subfertility.

Physiology of some actinomycete genera.

Actinomycetes are widespread in the environment and are mainly organotrophic. Studies of their ecology have been primarily focussed on their detection and isolation, with comparatively little attention to the control mechanisms that determine their occurrence and behaviour in their natural environments. This session provided some diverse examples of approaches to this problem. Several actinomycete genera produce motile spores. The significance of flagella proteins and factors influencing spore motility and germination are considered. The genus Frankia forms nitrogen-fixing associations with non-leguminous plants. Molecular techniques have been used to clarify the endophyte-host relationships. Micromonospora species are common in the environment. The growth and physiology of a gentamicin-producing strain are described. Thermophilic actinomycetes in the genus Thermoactinomyces are common in composts and other self-heating environments. Novel isolates from acid soil, which grow and produce enzymes active at high temperatures and in acidic conditions, are discussed.

The significance of reactions to betaxolol reported by patients.

Some patients may experience respiratory side effects on betaxolol in spite of the greater safety which is claimed for this ocular hypotensive drug compared with timolol. Six of 29 patients using betaxolol complained of wheeze or respiratory distress and five of these patients were rechallenged with betaxolol and a placebo in a double-masked clinical study, respiratory function being measured before and after each medication according to the American Thoracic Society guidelines. No patient showed any change in respiratory function either with betaxolol or the placebo. The findings further support the safety of betaxolol even in patients with respiratory disease, though some caution should be observed.

Systemic bacillus-Calmette Guerin infection after transurethral administration for superficial bladder carcinoma.

It is well known that intravesical bacillus Calmette-Guerin therapy is a safe and effective treatment for superficial bladder carcinoma with few serious complications. However, we report a case of pneumonitis, hepatitis, azotemia, fever and pancytopenia in a patient after transurethral administration of bacillus Calmette-Guerin. Even though severe systemic complications are rare, patients who have high fevers of signs of other systemic manifestations require documentation of the extent of the infection. Also, we address the use of prophylactic antituberculous drugs, not only to prevent these symptoms but also to raise the questions of diminishing the immunological effect of the treatment.