A tractable covalent linker strategy for the production of immunogenic antigen-TLR7/8L bioconjugates.

Despite the ease of production and improved safety profiles of recombinant vaccines, the inherently low immunogenicity of unadjuvanted proteins remains an impediment to their widespread adoption. The covalent tethering of TLR agonists to antigenic proteins offers a unique approach to co-deliver both constituents to the same cell-enhancing vaccine efficacy while minimizing reactogenicity. However, the paucity of simple and effective linker chemistries continues to hamper progress. Here, we present a modular, PEG-based linker system compatible with even extremely lipophilic and challenging TLR7/8 agonists. To advance the field and address previous obstacles, we offer the most straightforward and antigen-preserving linker system to date. These antigen-adjuvant conjugates enhance antigen-specific immune responses in mice, demonstrating the power of our approach within the context of modern vaccinology.

Non-firearm-related homicide, New Mexico, 2001-3.

OBJECTIVE: New Mexico (NM) has the highest rate of non-firearm-related homicide in the USA and ranks 20th in firearm-related homicides. Because non-firearm-related homicides are inadequately described in the literature, characterisation of non-firearm-related homicide victims will enhance efforts to reduce homicides. METHODS: Homicide victims were identified through the Office of the Medical Investigator. Age-specific and age-adjusted homicide death rates were calculated for 2001-3 by sex and race/ethnicity, and associations between covariates and non-firearm-related homicide were measured. RESULTS: Non-firearm-related homicides comprised 33% of US homicide victims, 47% of NM homicide victims, and 74% of NM American Indian (AI) homicide victims. Of 212 NM non-firearm-related homicide victims, 37% had been beaten, 32% had been stabbed, and 12% had been strangled. Females comprised 30% of non-firearm-related homicide victims and 18% of firearm-related homicide victims. A blood alcohol concentration (BAC) >or=0.08 mg/dl was detected among 43% of non-firearm-related (61% of AI) and 33% of firearm-related (50% of AI) homicide victims. Non-firearm-related homicide rates were highest among AI men aged 25-34 years (31/100,000). Non-firearm-related homicide victims were more likely than firearm-related victims to be AI (adjusted odds ratio (AOR) 4.20; 95% CI 2.16 to 8.16) and female (AOR 2.05; 95% CI 1.27 to 3.31), and to have had a BAC >or=0.08 mg/dl (AOR 1.65; 95% CI 1.08 to 2.52). CONCLUSIONS: Homicide-prevention efforts among AIs in NM should focus on non-firearm-related homicides. The association between excessive drinking and non-firearm-related homicide should be further characterised. Continued surveillance for non-firearm-related homicides will assist these efforts.

IgA nephropathy in the triethnic population of New Mexico.

AIMS: IgA nephropathy (IgAN) is the most frequent glomerulonephritis around the globe, but its incidence in the United States is unknown. The disease has a preponderance for certain racial/ethnic groups. Our goals were to retrospectively analyze a series of IgAN biopsies from the state of New Mexico and to calculate an estimated incidence. Then we compared the racial/ethnic composition of our patient cohort to the composition of the New Mexico population and examined the three main racial/ethnic groups for differences in clinical and pathologic parameters. MATERIALS AND METHODS: Renal biopsies and clinical data from IgAN cases newly diagnosed in New Mexico between 2000 and 2005 were reviewed. We compared the racial/ethnic composition of our patient cohort to the demographic composition of the New Mexico population. Demographic, clinical, and histopathologic variables were analyzed with respect to the patients' race/ethnicity. RESULTS: The incidence of IgAN in New Mexico was 10.2 cases per million persons per year (9.3 when Henoch-Schönlein purpura cases were excluded). American Indians were twice as frequent in our patient cohort when compared to their demographic representation, with the reverse finding for Non-Hispanic Whites. Hispanics more frequently had nephrotic range proteinuria than Non-Hispanic Whites and American Indians. On renal biopsy, endocapillary proliferative glomerulonephritis was the most common glomerular abnormality, followed by the focal segmental glomerulosclerosis (FSGS)-like pattern. The FSGS-like pattern was more frequent in American Indians and Hispanics than in Non-Hispanic Whites. CONCLUSIONS: This is the first report of an incidence figure of IgAN for an entire state in the US. American Indian and Hispanic patients had a stronger representation in our cohort than Non-Hispanic Whites, when compared to the general New Mexico population.

Cross-protection studies between respiratory and calf diarrhea and winter dysentery coronavirus strains in calves and RT-PCR and nested PCR for their detection.

A 1-step RT-PCR assay, targeting a 730 bp fragment of the nucleocapsid (N) gene of bovine coronavirus (BCV), and a nested PCR assay, targeting a 407 bp fragment of the N gene, were developed to detect BCV in nasal swab and fecal samples of calves experimentally exposed to BCV. Both 1-step RT-PCR and nested PCR recognized cell culture passaged isolates of 10 bovine respiratory coronavirus (BRCV), 5 calf diarrhea (CD) and 8 winter dysentery (WD) strains of BCV, but not transmissible gastroenteritis coronavirus or bovine rotavirus. The sensitivity of the 1-step RT-PCR and nested PCR was compared to that of an antigen-capture ELISA. The lowest detection limit of the 1-step RT-PCR and nested PCR as determined by using tenfold serial dilutions of the BRCV 255 and 440 strains in BCV negative nasal swab suspensions from preexposure gnotobiotic calves was 2 x 10(4) and 2 x 10(2) TCID50/0.1 ml for each strain, respectively. The lowest detection limit of the antigen-capture ELISA as determined by using the same serially diluted samples was 1 x 10(6) TCID50/0.1 ml for each strain. Therefore, the 1-step RT-PCR and nested PCR assays were 50 and 5000 times, respectively more sensitive than the antigen-capture ELISA to detect BRCV in nasal swab suspensions. To investigate in vivo cross-protection between the BRCV and CD or WD strains of BCV and to detect nasal and fecal shedding of BCV using the 1-step RT-PCR, nested PCR and antigen-capture ELISA, 6 colostrum-deprived and two gnotobiotic calves were inoculated with a BRCV, a CD or a WD strain of BCV and then challenged 3-4 weeks later with either BRCV, CD or WD strains of BCV. All calves developed diarrhea after inoculation and BCV antigen (ELISA) or RNA (RT-PCR) was detected in the diarrheic fecal samples or the corresponding nasal swab samples. In addition, low amounts of BCV were also detected only by nested PCR in the fecal and nasal swab samples before and after diarrhea. No respiratory clinical signs were observed during the entire experimental period, but elevated rectal temperatures were detected during diarrhea in the BCV-inoculated calves. All calves recovered from infection with the BRCV, CD, or WD strains of BCV were protected from BCV-associated diarrhea after challenge exposure with either a heterologous or homologous strain of BCV. However, all calves challenged with heterologous BCV strains showed subclinical BCV infection evident by detection of nasal and fecal shedding of BCV RNA detected only by nested PCR. Such results confirm field and experimental data documenting reinfection of the respiratory and enteric tracts of cattle, suggesting that, in closed herds, respiratory or enteric tract reinfections may constitute a source of BCV transmissible to cows (WD) or neonatal or feedlot calves. In addition, the present 1-step RT-PCR and nested PCR assays were highly sensitive to detect BCV in nasal swab and fecal specimens. Therefore, these assays should be useful to diagnose BCV infections in calves and adult cows.

An outbreak of primary pneumonic tularemia on Martha's Vineyard.

BACKGROUND: In the summer of 2000, an outbreak of primary pneumonic tularemia occurred on Martha's Vineyard, Massachusetts. The only previously reported outbreak of pneumonic tularemia in the United States also occurred on the island in 1978. METHODS: We conducted a case-control study of adults with pneumonic tularemia and investigated the environment to identify risk factors for primary pneumonic tularemia. Patients with confirmed cases were residents of or visitors to Martha's Vineyard who had symptoms suggestive of primary pneumonic tularemia, were ill between May 15 and October 31, 2000, and had a positive laboratory test for tularemia. Controls were adults who had spent at least 15 days on Martha's Vineyard between May 15 and September 28, 2000. RESULTS: We identified 15 patients with tularemia; 11 of these cases were primary pneumonic tularemia. Francisella tularensis type A was isolated from blood and lung tissue of the one man who died. Patients were more likely than controls to have used a lawn mower or brush cutter in the two weeks before the illness or before an interview, for controls (odds ratio, 9.2; 95 percent confidence interval, 1.6 to 68.0) and during the summer (odds ratio, undefined; 95 percent confidence interval, 1.8 to infinity). Lawn mowing and brush cutting remained significant risk factors after adjustment for other potentially confounding variables. Only one patient reported being exposed to a rabbit while cutting brush. Of 40 trapped animals, 1 striped skunk (Mephitis mephitis) and 1 Norway rat (Rattus norvegicus) were seropositive for antibodies against F. tularensis. CONCLUSIONS: Study of this outbreak of primary pneumonic tularemia implicates lawn mowing and brush cutting as risk factors for this infection.

Antibody titers against bovine coronavirus and shedding of the virus via the respiratory tract in feedlot cattle.

OBJECTIVE: To describe patterns of seroconversion to bovine coronavirus (BCV) and shedding of BCV from the respiratory tract in feedlot cattle. ANIMALS: 1,074 calves in feedlots in Ohio, Texas, and Nebraska. PROCEDURE: Nasal swab specimens were obtained at time of arrival (day 0) and at various times during the initial 28 days after arrival at feedlots. Specimens were tested for BCV, using an antigen-capture ELISA. Serum samples were obtained at time of arrival and again 28 days after arrival; sera were analyzed for antibodies to BCV, using an antibody-detection ELISA. RESULTS: Samples from 12 groups of cattle entering 7 feedlots during a 3-year period revealed that 78 of 1,074 (7.3%) cattle were shedding BCV (range, 0 to 35.9% within specific groups). At time of arrival, 508 of 814 (62.4%) cattle had low (< 50) or undetectable BCV antibody titers. Seroconversion to BCV during the initial 28 days after arrival was detected in 473 of 814 (58%) cattle tested (range, 20.3 to 84.1 % within specific groups). In cattle shedding BCV from the nasal passages, 49 of 68 (72.1 %) seroconverted, and 472 of 746 (63.3%) cattle that were not shedding the virus seroconverted. CONCLUSIONS AND CLINICAL RELEVANCE: Bovine coronavirus can be detected in populations of feedlot cattle in the form of viral shedding as well as seroconversion to the virus. Although only a few cattle were shedding the virus at the time of arrival at a feedlot, most of the cattle seroconverted to BCV by 28 days after arrival.

Association between infection of the respiratory tract attributable to bovine coronavirus and health and growth performance of cattle in feedlots.

OBJECTIVE: To determine the association between respiratory tract infection with bovine coronavirus (BCV), treatment for respiratory tract disease, pulmonary lesions at slaughter, and average daily gain in cattle in feedlots. ANIMALS: 837 calves in feedlots in Ohio and Texas. PROCEDURE: Nasal swab specimens were obtained from cattle at arrival in a feedlot (day 0) and at various times during the initial 28 days after arrival. Specimens were tested for BCV, using an antigen-capture ELISA. Serum samples were obtained at arrival and again 28 days after arrival and tested for antibodies to BCV, using an antibody-detection ELISA. Information was collected regarding treatment for cattle with respiratory tract disease and average daily gain during the feeding period. Pulmonary lesions were evaluated at slaughter. RESULTS: Cattle shedding BCV from the nasal cavity and developing an antibody response against BCV were 1.6 times more likely to require treatment for respiratory tract disease than cattle that did not shed the virus or develop an immune response against BCV. Additionally, cattle that shed BCV from the nasal cavity were 2.2 times more likely to have pulmonary lesions at slaughter than cattle that did not shed the virus. The BCV shedding or seroconversion status did not affect average daily gain. CONCLUSIONS AND CLINICAL RELEVANCE: Bovine coronavirus infects feedlot cattle and is associated with an increased risk for cattle developing respiratory tract disease and pulmonary lesions. Development of appropriate control measures could help reduce the incidence of respiratory tract disease.

Chlamydia-dependent biosynthesis of a heparan sulphate-like compound in eukaryotic cells.

One hypothesis for the mechanism of chlamydial interaction with its eukaryotic host cell invokes a trimolecular mechanism, whereby a Chlamydia-derived glycosaminoglycan bridges a chlamydial acceptor molecule and a host receptor enabling attachment and invasion. We show that a heparan sulphate-specific monoclonal antibody specifically binds a glycosa-minoglycan localized to the surface of the chlamydial organism and effectively neutralizes infectivity of both C. trachomatis and C. pneumoniae. In addition to the ability of this antibody to neutralize infectivity, direct visualization using immunofluorescence demonstrated staining of chlamydial organisms localized to the intracellular vacuole. The chlamydial-associated glycosaminoglycan was specifically labelled with [14C]-glucosamine, and the labelled compound was immunoprecipitated and resolved by gel electrophoresis. The chlamydial-associated glycosaminoglycan is a high-molecular-weight compound similar in size to heparin or heparan sulphate and was sensitive to cleavage by heparan sulphate lyase. These data demonstrate that a glucosamine-containing sulphated polysaccharide is produced within the intracellular vacuole containing chlamydiae and is a target for antibody-mediated neutralization of infectivity.

Resting rectal temperature of Vietnamese potbellied pigs.

OBJECTIVE: To determine resting rectal temperatures of Vietnamese potbellied pigs. DESIGN: Prospective clinical trial. ANIMALS: 85 potbellied pigs on a single farm and 27 potbellied pigs examined at a veterinary teaching hospital for routine veterinary care. PROCEDURE: Rectal temperatures of the potbellied pigs on a farm were measured during the morning, afternoon, and evening. Rectal temperatures at the time of initial examination were obtained from medical records for the potbellied pigs examined at the hospital. RESULTS: Mean rectal temperatures for both groups of potbellied pigs were the same. Overall unadjusted mean +/- SD rectal temperature was 37.6 +/- 0.8 C (99.7 +/- 1.5 F; range, 35.1 to 39.6 C [95.2 to 103.3 F]). However, diurnal variation in rectal temperature was found among the farm population of potbellied pigs. After adjustment for age and repeated sampling, rectal temperatures recorded during the morning were found to be significantly lower than temperatures recorded during the afternoon and evening (mean difference, 0.5 and 0.9 C [0.9 and 1.6 F], respectively), and rectal temperatures recorded during the afternoon were found to be significantly lower than temperatures recorded during the evening (mean difference, 0.4 C [0.7 F]). There was a significant inverse linear relationship between age and rectal temperature. CONCLUSIONS AND CLINICAL RELEVANCE: Rectal temperatures of Vietnamese potbellied pigs may be lower than the lower limit of the reference range reported for domestic pigs. Because of diurnal variation in rectal temperatures, it is important to compare temperatures obtained at the same time of day when assessing patients.

Antigenic variation among bovine enteric coronaviruses (BECV) and bovine respiratory coronaviruses (BRCV) detected using monoclonal antibodies.

Bovine coronavirus (BCV) causes neonatal calf diarrhea (CD) and is associated with winter dysentery (WD) in adult dairy cattle. It can also be isolated from the respiratory tracts of cattle entering feedlots. Monoclonal antibodies (MAbs) specific for the hemagglutinin esterase (HE) and spike (S) surface proteins of 2 bovine enteric coronavirus (BECV) strains and two bovine respiratory coronavirus (BRCV) strains were tested against 6 BECV strains and 6 recently isolated BRCV strains, in order to characterize the antigenicity of BCV strains with varied tissue tropisms. All MAbs had high immunofluorescence (IF) titers against BECV and BRCV strains, indicative of conserved cross-reactive epitopes. In hemagglutination inhibition (HI) tests, the S-MAbs were more broadly reactive than HE-MAbs. The BRCV and CD MAbs were more broadly reactive in HI than the WD MAbs. The HA activity of the Mebus vaccine CD strain was not inhibited by any of the MAbs tested. The HI activity of BRCV strain R6 was unique among the 6 BRCV isolates. In virus neutralization assays, MAbs to the BRCV strain R4 neutralized all 6 BECV strains tested. Antigenic variation exists among both BECV and BRCV strains, but it cannot be attributed soley to the clinical origin of the strain.

Isolation of bovine respiratory coronaviruses from feedlot cattle and comparison of their biological and antigenic properties with bovine enteric coronaviruses.

OBJECTIVE: To isolate bovine coronaviruses from the respiratory tracts of feedlot cattle and compare antigenic and biological properties of these strains with bovine enteric coronaviruses. ANIMALS: 5- to 8-month-old mixed-breed cattle at 4 feedlots. PROCEDURE: Samples were obtained from the nasal passages for testing. The 13 samples with the highest magnitude of positive values for bovine coronavirus (BCV) were cultured. Ten strains of bovine respiratory coronavirus (BRCV) were adapted successfully to serial passage. After observation of cytopathic effects (CPE) and confirmation of BRCV by immune electron microscopy and immunofluorescence testing, cell culture-adapted strains were cloned by limiting dilution. These isolates then were compared with a panel of bovine enteric coronaviruses (BECV), using hemagglutination (HA), receptor-destroying enzyme activity (RDE), hemagglutination inhibition (HI), and virus neutralization (VN) assays. Antigenic relatedness values then were calculated. RESULTS: The BRCV were detected in 105 of 488 (21.5%) of the cattle tested. Of 13 strains tested, 10 were isolated in cell culture. Six of the BRCV strains were similar to 2 strains obtained from neonatal calves with diarrhea and 2 strains from adult cattle with winter dysentery. The other 4 BRCV isolates had high RDE activity against mouse erythrocytes but differed from other strains of BECV Nine of 10 BRCV isolates had properties similar to the 2 BECV subtypes. CONCLUSIONS AND CLINICAL RELEVANCE: The BRCV can be isolated from nasal passages of cattle entering feedlots. Most BRCV were similar to BECV strains, although a few had unique properties. Vaccines developed to protect against enteric strains also may protect against respiratory tract strains.

The Iowa hospital visitation program: does outreach education affect management of neonatal resuscitation?

Thirty-nine Iowa hospitals were visited by a perinatal education team to improve management of neonatal resuscitation. Patient care was evaluated by reviewing medical records according to pre-established performance standards. Following patient-care review, educational programs were offered to physicians and nurses. The hospitals were first visited from July 1982 until June 1984. Follow-up visits were made during the next two years. Management of neonates born in meconium was initially inadequate, but improved during the second period. Similarly, the conduct of other neonatal care practices showed statistically significant improvement over time (including assisted ventilation, hypoglycemia screening, cardiac massage, temperature maintenance, and record documentation). Only treatment of hypoglycemia failed to show significant change. We discuss why a cause and effect relationship cannot be claimed even though the desired effect of the educational intervention was achieved.

Partial exchange transfusion in term, polycythemic neonates: absence of association with severe gastrointestinal injury.

Partial exchange transfusion is often used to treat neonatal polycythemia. Concern about the risk of necrotizing enterocolitis following the procedure has recently been raised. We report a retrospective analysis of 185 term, polycythemic neonates who received partial exchange transfusion. No evidence of severe gastrointestinal injury was found. The technique for partial exchange transfusion we report consists of removal of blood from the umbilical vein with reinfusion of a commercial plasma substitute through a peripheral vein. Based on our data and a literature review, we offer suggestions for future conduct of partial exchange transfusion in polycythemic neonates.

The changing pattern of neonatal mortality in a regionalized system of perinatal care.

Neonatal deaths in Iowa were reviewed for the years 1982 and 1983. As in a similar review of deaths in 1978 and 1979, respiratory distress syndrome, bacterial sepsis, asphyxia, lethal malformations, and extreme immaturity accounted for approximately 90% of deaths. Fewer deaths occurred in 1982 and 1983 and the causes of death shifted toward the nonpreventable. Lethal malformations became the leading cause of death and showed an increased incidence over the previous period. The greatest reduction of deaths was in level 1 hospitals. Ability to effect further reduction in neonatal deaths was estimated by calculation of an idealized neonatal mortality rate for the state and each level of care. These calculations suggest that future reduction in mortality must come primarily from improved care in level 2 and 3 centers rather than from further change in level 1 provider behavior.

Comparing perinatal mortality.

Comparisons of perinatal outcome data among regions or hospitals can be misleading if the risk status of the population served is not considered. Data are presented from two large perinatal centers (Medical Center Hospital in San Antonio, Texas, and University of Iowa Hospitals in Iowa City, Iowa), which report substantially different institutional neonatal and fetal mortality rates. However, examination of neonatal and fetal mortality by birth weight groupings demonstrates the difference between outcome in the two hospitals to be considerably less than the overall rates might imply. Additional data about the regionalized system of care in Iowa are presented to illustrate why mortality may rise in a referral center and yet be consistent with a salutary effect on perinatal outcome in the region.