Altered Platelet-Megakaryocyte Endocytosis and Trafficking of Albumin and Fibrinogen in RUNX1 Haplodeficiency.

Platelet α-granules have numerous proteins, some synthesized by megakaryocytes (MK) and others not synthesized but incorporated by endocytosis, an incompletely understood process in platelets/MK. Germline RUNX1 haplodeficiency, referred to as familial platelet defect with predisposition to myeloid malignancies (FPDMM), is associated with thrombocytopenia, platelet dysfunction and granule deficiencies. In previous studies, we found that platelet albumin, fibrinogen and IgG levels were decreased in a FPDMM patient. We now show that platelet endocytosis of fluorescent-labeled albumin, fibrinogen and IgG is decreased in the patient and his daughter with FPDMM. In megakaryocytic human erythroleukemia (HEL) cells, siRNA RUNX1 knockdown (KD) increased uptake of these proteins over 24 hours compared to control cells, with increases in caveolin-1 and flotillin-1 (two independent regulators of clathrin-independent endocytosis), LAMP2 (a lysosomal marker), RAB11 (a marker of recycling endosomes) and IFITM3. Caveolin-1 downregulation in RUNX1-deficient HEL cells abrogated the increased uptake of albumin, but not fibrinogen. Albumin, but not fibrinogen, partially colocalized with caveolin-1. RUNX1 knockdown increased colocalization of albumin with flotillin and of fibrinogen with RAB11 suggesting altered trafficking of both. The increased albumin and fibrinogen uptake and levels of caveolin-1, flotillin-1, LAMP2 and IFITM3 were recapitulated by shRNA RUNX1 knockdown in CD34 + -derived MK. These studies provide the first evidence that in RUNX1- haplodeficiency platelet endocytosis of albumin and fibrinogen is impaired and that megakaryocytes have enhanced endocytosis with defective trafficking leading to loss of these proteins by distinct mechanisms. They provide new insights into mechanisms governing endocytosis and α-granule deficiencies in RUNX1- haplodeficiency. Key points: Platelet content and endocytosis of α-granule proteins, albumin, fibrinogen and IgG, are decreased in germline RUNX1 haplodeficiency. In RUNX1 -deficient HEL cells and primary MK endocytosis is enhanced with defective trafficking leading to decreased protein levels.

Mite dispersal among the Southern Ocean Islands and Antarctica before the last glacial maximum.

It has long been maintained that the majority of terrestrial Antarctic species are relatively recent, post last glacial maximum, arrivals with perhaps a few microbial or protozoan taxa being substantially older. Recent studies have questioned this 'recolonization hypothesis', though the range of taxa examined has been limited. Here, we present the first large-scale study for mites, one of two dominant terrestrial arthropod groups in the region. Specifically, we provide a broad-scale molecular phylogeny of a biologically significant group of ameronothroid mites from across the maritime and sub-Antarctic regions. Applying different dating approaches, we show that divergences among the ameronothroid mite genera Podacarus, Alaskozetes and Halozetes significantly predate the Pleistocene and provide evidence of independent dispersals across the Antarctic Polar Front. Our data add to a growing body of evidence demonstrating that many taxa have survived glaciation of the Antarctic continent and the sub-Antarctic islands. Moreover, they also provide evidence of a relatively uncommon trend of dispersals from islands to continental mainlands. Within the ameronothroid mites, two distinct clades with specific habitat preferences (marine intertidal versus terrestrial/supralittoral) exist, supporting a model of within-habitat speciation rather than colonization from marine refugia to terrestrial habitats. The present results provide additional impetus for a search for terrestrial refugia in an area previously thought to have lacked ice-free ground during glacial maxima.

Genetic epidemiology of Darier's disease: a population study in the west of Scotland.

BACKGROUND: Darier's disease has a world-wide distribution, but estimates of prevalence have varied. The discovery that the disease is due to mutations in ATP2A2 provides the opportunity to study the genetic epidemiology of the disease in localized populations. OBJECTIVES: To survey the prevalence of Darier's disease in the west of Scotland and look for founder effects in this population. METHODS: We ascertained cases of Darier's disease in the west of Scotland and used genealogy and mutational analysis to seek common ancestry. RESULTS: Seventy-eight current cases were identified, giving a prevalence of approximately 1 : 30 000. While 63 cases gave a history of Darier's disease in previous generations, conventional genealogy identified only two pairs of two family groups with common ancestry within the last 180 years. Eleven patients (14%; three of whom had in total four affected children) had probable de novo mutations. Causative mutations in ATP2A2 have been identified in 11 of 15 pedigrees screened for mutation, but no two share the same mutation. CONCLUSIONS: High estimates of prevalence are likely to be due to intensive ascertainment, rather than founder effects. Darier's disease is likely to be more common than has been recognized in other populations.

Thirty-five year mortality following receipt of SV40- contaminated polio vaccine during the neonatal period.

Early poliovirus vaccines, both inactivated and live attenuated, were inadvertently contaminated with simian virus 40 (SV40), a monkey virus known to be oncogenic for newborn hamsters. Although large epidemiologic studies have not identified an elevated cancer risk in persons who received SV40-contaminated vaccines, fragments of SV40 DNA have recently been identified in certain human tumours. We report the follow-up of a cohort of 1073 persons, unique because they received SV40-contaminated poliovirus vaccines as newborns in 1961-63. A previous report of the status of these subjects as of 1977-79 identified 15 deaths, none due to cancer. The present study utilized the National Death Index to identify deaths in the cohort for the years 1979-96. Expected deaths were calculated from Cleveland area sex-, age-, race- and year-specific mortality rates. Increased mortality from all causes was not found. 4 deaths from cancer were found compared to 3.16 expected (P = 0.77). However, 2 deaths from testicular cancer occurred, compared to 0.05 expected (P = 0.002), which may be a chance finding due to multiple comparisons. There were 2 deaths due to leukaemia, a non-significant finding, and no deaths due to tumours of the types putatively associated with SV40. Although these results are, for the most part, consistent with other negative epidemiologic investigations of risks from SV40-contaminated vaccines, further study of testicular cancer may be warranted, and it will be important to continue monitoring this cohort which is now reaching middle-age.

Sudden infant death syndrome, bedsharing, parental weight, and age at death.

OBJECTIVE: To assess the role of parental bedsharing in sudden infant death syndrome (SIDS)-like deaths, this study examines the hypothesis that, compared with other SIDS cases, the age distribution of deaths associated with bedsharing should be lower in younger, less vigorous infants. METHODS: For 84 SIDS cases in Cleveland, Ohio, 1992 to 1996, age at death, maternal weight, and other risk factors for SIDS were compared for cases grouped according to bedsharing status. RESULTS: Mean ages at death were 9.1 weeks for 30 bedsharing and 12.7 for 54 nonbedsharing cases, counting 10 with missing information as nonbedsharing. Mean pregravid weights of bedsharing mothers exceeded those of nonbedsharing mothers (84.1 vs 67.0 kg). Mean ages at death for nonbedsharing infants, bedsharing infants of smaller mothers, and bedsharing infants of larger mothers were 12.7, 10.3, and 7.6 weeks, respectively. Large maternal size did not affect age at death in the absence of bedsharing. CONCLUSIONS: By demonstrating that among an urban population at high risk for SIDS, bedsharing is strongly associated with a younger age at death, independent of any other factors, this study provides evidence of a relationship between some SIDS-like deaths and parent-infant bedsharing, particularly if the parent is large.

Antibody responses to Bordetella pertussis antigens and clinical correlations in elderly community residents.

A serological study to determine the frequency of Bordetella pertussis infection in 100 adults aged >/=65 years was carried out over a 3-year period. Ten serum samples (collected every 4 months) from each subject were examined for IgA and IgG antibodies to the following B. pertussis antigens: pertussis toxin (PT), filamentous hemagglutinin (FHA), pertactin, and fimbriae-2. A >/=2-fold titer increase in ELISA units from one time period to the next was considered serological evidence of infection. The rate of serologically defined infection (i.e., in which there was an increase in titer against any antigen) was 19.7 per 100 person-years. With the use of more specific criteria that indicate definite B. pertussis infection (>/=2-fold increase in titer to PT) and probable B. pertussis infection (>/=2-fold increase in titer to PT or >/=2-fold increase to fimbriae-2), the rates were 3.3 and 8.0 per 100 person-years, respectively. Fifty percent of individuals with definite B. pertussis infections had time-associated symptomatology. Antibody patterns over time suggest that antibody to FHA and perhaps to pertactin is stimulated by infections with other organisms, as well as B. pertussis infections. Our data suggest that symptomatic pertussis occurs in elderly individuals. Consideration should be given to immunization of the elderly with acellular pertussis vaccines.

The effect of human immunodeficiency virus-1 infection and stunting on measles immunoglobulin-G levels in children vaccinated against measles in Uganda.

BACKGROUND: Measles is an important public health problem in developing nations and there is concern that immune response to measles vaccination may be compromised by paediatric human immunodeficiency virus (HIV) infection. Therefore, this study investigated the relationship of immunoglobulin-G (Ig-G) antibody levels in children vaccinated against measles and HIV-1 infection. Further analysis was done to assess the influence of nutritional status on this relationship. METHODS: The authors measured HIV and measles antibodies in 243 vaccinated children aged 17-41 months from Kampala, Uganda. Children were from paediatric and HIV clinics. Potential confounders of this relationship included nutritional anthropometric measures, age at and time since vaccination, measles exposure, family crowding, vaccination clinic and gender. Univariable and multivariable analyses were used to study these associations. RESULTS: Fifty children (21%) were HIV infected. In univariable analysis, low measles antibody (<15 ELISA units/ml) was associated with HIV (P = 0.05; odds ratio (OR) = 1.86) and stunting (P = 0.06; OR = 1.68). Stunting, measured as height-for-age and defined as <-2 standard deviations of the reference population median, was a surrogate for chronic malnutrition. HIV was strongly associated with stunting (P = 0.0001; OR = 6.62). In multiple logistic regression, HIV was not associated with low measles antibodies; however, stunting (P = 0.04; OR = 1.81), and <3 children in the home (P = 0.01; OR = 1.96) were. Conversely, being male (P = 0.05; OR = 0.58), and measles in the home in the previous month (P = 0.04; OR = 0.33) were associated with high antibody levels. CONCLUSIONS: These findings suggest that HIV in Ugandan children immunized for measles may indirectly interfere with measles antibody levels by causing malnutrition, the latter being the direct cause. Programmes to improve childhood nutrition, especially for HIV-infected children, should accompany measles control programmes in developing nations for maximum benefit from measles vaccination. HIV infection per se should not change measles vaccination programmes.

PIP: The effect of HIV infection upon measles immunity was explored in children vaccinated against measles. HIV and measles antibody levels were measured in 243 vaccinated children aged 17-41 months from pediatric and HIV clinics in Kampala, Uganda. Potential confounders of the relationship included nutritional anthropometric measures, age at and time since vaccination, measles exposure, family crowding, vaccination clinic, and gender. 50 children (21%) were infected with HIV. Multiple logistic regression analysis found HIV to not be associated with a low level of measles antibodies, but with stunting and having less than 3 children in the home. Being male and having measles in the home during the previous month were associated with high antibody levels. These findings suggest that HIV in Ugandan children immunized for measles may indirectly interfere with measles antibody levels by causing malnutrition. Programs to improve childhood nutrition, especially for HIV-infected children, should therefore accompany measles control programs in developing countries in order to reap the maximum benefit from measles vaccination.

Effect of rotation on periprosthetic bone mineral measurements in a hip phantom.

The effect of rotation on the measurement of periprosthetic bone mineral content and bone mineral density using dual energy xray absorptiometry was studied in 9 anatomic specimen femora implanted with noncemented titanium hip prostheses. An apparatus was constructed to permit accurate femoral rotation through an arc of 30 degrees internal to 30 degrees external rotation in 5 degrees increments. Two scans were obtained at each increment of rotation for a total of 26 scans for each implanted femur. Change in bone mineral content and bone mineral density was measured for each of 7 contiguous periimplant regions (Gruen zones). The precision of the paired bone mineral density measurements was 1.7%. In all Gruen zones, the means of bone mineral content and bone mineral density measurements varied within 5% between 15 degrees internal and 15 degrees external rotation. Variation in limb rotation, as might occur with temporal studies of periimplant bone remodeling, can be tolerated within easily controllable limits without excessive error in bone mineral measurements.

The university/public health link in reducing infant mortality in a high-risk population.

The Greater Cleveland Healthy Family/Healthy Start (HF/HS) Project Consortium was established to combine various resources in the Cleveland community in an effort to reduce the infant mortality rate by 50% over five years. As part of HF/HS, an Infant Mortality Review Program (IMRP) was instituted. This program has two important facets: an in-depth review of the circumstances of each death, and the development of scientifically-based, community-oriented interventions based on these reviews. To conduct the IMRP, a "hot line" connection with each hospital and the coroner's office was established to provide prompt notification of infant deaths and to enable timely data collection. A core review group determines actual cause of death and preventability, and identifies appropriate interventions. Recommendations issued by a technical oversight panel are disseminated to groups with the authority to institute changes in an effort to foster continuous quality improvement. Complete reviews of 243 of 319 deaths reported to date have resulted in 11 recommendations for presentation to community groups. The public health department's cooperation with other agencies has facilitated a process which ensures timely implementation. Cleveland's strongly committed consortium was able to link university and public health expertise to activate community groups and health care providers to implement recommended solutions. Demonstration of a measurable decline in the community's infant mortality rate (IMR) should provide concrete evidence of the value of such linkages. We suggest that a similar review mechanism be considered for other communities with high infant mortality rates.

Acute respiratory illness in older community residents.

OBJECTIVE: To describe the 3-year incidence of acute respiratory illness in a population of noninstitutionalized elderly persons. DESIGN: Cohort analytic study with an observation period of 3 years. SETTING: Large midwestern urban community. SUBJECTS: 574 noninstitutionalized persons aged 65 years and older, of whom 349 were living independently, 110 were in congregate settings, and 115 were living independently and regularly caring for small children. METHODS: Subjects were interviewed and examined by nurse practitioners at intake and every 4 months. Acute illness and convalescent visits were made when illnesses were reported by participants. MAIN OUTCOME MEASURES: The incidence of acute respiratory illness was used to test the hypothesis that elderly persons taking care of children have the highest incidence, congregate-dwellers intermediate incidence, and those living independently the lowest incidence. RESULTS: The average incidence of acute respiratory illness was 2.5 per 100 person months, comparable to rates reported in the National Health Survey. The incidence of respiratory illness was significantly greater in subjects living in congregate settings or regularly caring for young children. Similarly, greater proportions of persons in the congregate and child-care groups reported at least one episode of illness (P < .05). A more detailed analysis of exposure shows that acute respiratory illness was significantly more common in subjects who had contact with children (P < .05). This risk was definitely present during the months November through February, and probably also during July through October, but was not observed for March through June. CONCLUSIONS: Elderly persons living in the community experience low rates of acute upper respiratory illness. Contact with children increases their risk of developing such illnesses during certain seasons of the year.

Unrecognized pertussis infection in adolescents.

Little information is available regarding the level of immunity to Bordetella pertussis among adolescents. We measured serum antibodies in 156 healthy adolescents to the following pertussis antigens: pertussis toxin, filamentous hemagglutinin, and 69-kd outer membrane protein. In an attempt to identify intercurrent pertussis infections, we also obtained a total of 43 repeated samples during the following 5 years. Using a 50% or greater rise in IgG enzyme-linked immunosorbent assay titers to define seroconversion, we found an annual incidence of 6.1%; by alternative definitions of seropositivity, the predicted annual incidence of infection ranged from 1.2% to 8.2%. These data suggest that infection with B pertussis is common in the adolescent population.

Colonization and cross-colonization of nursing home patients with trimethoprim-resistant gram-negative bacilli.

A prospective study of 67 patients in a nursing home and a subpopulation of 31 patients who were stratified according to functional level--a general measure of the amount of nursing care required by the patients--was performed. The goals of this study were (1) to determine whether antibiotic selection of resistant variants from within endogenous flora of hosts or horizontal transmission is more important in the development of colonization with trimethoprim-resistant gram-negative bacilli (TRGNB) in a nursing home; (2) to identify the mode(s) of transmission if horizontal transmission is more important; and (3) to identify risk factors for colonization and cross-colonization with TRGNB. Although a number of variables were associated with colonization, only a decreased functional level appeared to be independently associated with colonization. Isolates from the subpopulation were subtyped by restriction endonuclease digestion of cellular DNA, as were isolates from personnel of the nursing home. Results revealed that 16 of 21 staff members had 48 positive cultures for TRGNB. Of 25 typeable isolates, 12 from seven of the 21 staff members were identical to patient strains. Analysis of acquisition of new strains of TRGNB by members of the subpopulation showed that 67.5% were the result of cross-colonization. Our data are consistent with, but do not prove, the hypothesis that nurses' hands are the primary mode of transmission of TRGNB in this nursing home and suggest that most colonization could be prevented by interdicting horizontal transmission.

When, where, and how do immunizations fail?

There are two main reasons for failure of immunizations: (1) failure of the vaccine delivery system to provide potent vaccines properly to persons in need; and (2) failure of the immune response, whether due to inadequacies of the vaccine or factors inherent in the host. The first category is by far the most important worldwide. The major factor contributing to failure of the delivery system is failure to vaccinate; in the developing world this is commonly a result of inadequacy of the vaccine supply. Other important factors include barriers to immunizations, improper use of vaccines, vaccine ineffectiveness at the time of use, and factors relating to client attitudes and knowledge. Failure of the immune response may be either primary or secondary (loss of protection after initial effectiveness). The shortcomings of existing vaccines must not deter us from taking maximal advantage of their benefits.

Epidemiology of pertussis and reactions to pertussis vaccine.

It remains clear that pertussis is a dangerous infectious disease that is well-controlled in industrialized countries by widespread immunization. In the developing world, it remains a source of high morbidity and mortality because of previously inadequate immunization programs. However, because of the intense efforts of the World Health Organization's Expanded Programme on Immunization, the effects of pertussis have already been ameliorated and show promise of being within a decade of approximating the situation in the developed world. Pertussis can be controlled only by immunization; other measures such as antimicrobial therapy offer negligible benefit. A problem that has been addressed in recent years is the excessive reactivity of whole-cell pertussis vaccine, which undoubtedly includes components of the organism that are irrelevant to the induction of immunity and are excessively reactive. Although epidemiologic studies appear to have largely, if not completely, absolved pertussis vaccine of responsibility for inducing death or permanent neurologic disability, a less reactive vaccine is highly desirable, not only to promote acceptance of a full course of immunization for the world's children but also for simple humanitarian reasons. Additionally, it has become evident that, because of waning immunity, pertussis increasingly occurs in adults. A less reactive vaccine would offer opportunity for reinforcement of immunity beyond childhood. The development of better, though as yet incomplete, understanding of the biology of Bordetella pertussis and its relation to humanity offers the opportunity for the production of less reactive vaccines free of irrelevant components. Acellular pertussis vaccines have been used exclusively in Japan for more than 10 years, and one such preparation, combined with diphtheria and tetanus toxoids, was licensed in the United States in late 1991 for use as the fourth and fifth doses of DTP, given at 15 months and prior to school entry. Field trials of this and other acellular DTP preparations are currently under way to determine their clinical efficacy in infants. It is probable that, within a very few years, whole-cell pertussis vaccine will be replaced by these newer preparations and that, in addition, the acellular product will be combined with other antigens, such as Haemophilus influenzae type b vaccine.

Producing and avoiding stress shielding. Laboratory and clinical observations of noncemented total hip arthroplasty.

Experimental canine model studies of stiff versus flexible, fully porous-coated, metallic femoral stems (differing by three- to fivefold in stiffness characteristics) revealed markedly different resorptive bone remodeling patterns. The flexible stem resulted in about 30% more cortical bone retention adjacent to the implant at one-year postimplantation and larger differences in dogs killed two and three years after surgery. Strain-gauge studies confirmed that there are differences in cortical bone strains with the two stem designs, the flexible stem producing a more uniform and more nearly normal strain distribution medially. Differences in cortical bone remodeling were quantified using dual energy X-ray absorptiometry (DEXA). The bone mineral content in femora with the flexible stem decreased less than 20%, compared to normal. At three years postimplantation, the bone mineral content of the femora with the stiff stem was about 50% that of the femora with the flexible stem. Clinically, DEXA revealed that 5%-15% changes in bone mineral density at various periimplant sites were common within the first two years after surgery; these changes were not usually evident roentgenographically. Serial roentgenographically distinct bone resorption was usually associated with bone mineral density changes of 20%-50%. Five- to 13-year roentgenographic follow-up observations of 213 cases with the Anatomic Medullary Locking prosthesis showed that pronounced bone resorption occurred in 33% of patients. Larger stems (greater than 13 mm in diameter) and stems with extensive porous coating had a significantly higher incidence of pronounced bone resorption than smaller stems and those with proximal coating. The stiffness characteristics of the human femur were established as a function of canal size and compared with those of noncemented hip prostheses. Increased mechanical compatibility was found for stems made of titanium alloy and with design features that reduce cross-sectional area and moment of inertia. Clinical data suggest that to reduce the likelihood of pronounced bone resorption, it would be beneficial for the implant to possess a bending stiffness of about one half to one third that of the human femur.