Therapeutic TNF Inhibitors Exhibit Differential Levels of Efficacy in Accelerating Cutaneous Wound Healing.

Adalimumab but neither etanercept nor certolizumab-pegol has been reported to induce a wound-healing profile in vitro by regulating macrophage differentiation and matrix metalloproteinase expression, which may underlie the differences in efficacy between various TNF-α inhibitors in impaired wound healing in patients with hidradenitis suppurativa, a chronic inflammatory skin disease. To examine and compare the efficacy of various TNF inhibitors in cutaneous wound healing in vivo, a human TNF knock-in Leprdb/db mouse model was established to model the impaired cutaneous wound healing as seen in hidradenitis suppurativa. The vehicle group exhibited severe impairments in cutaneous wound healing. In contrast, adalimumab significantly accelerated healing, confirmed by both histologic assessment and a unique healing transcriptional profile. Moreover, adalimumab and infliximab showed similar levels of efficacy, but golimumab was less effective, along with etanercept and certolizumab-pegol. In line with histologic assessments, proteomics analyses from healing wounds exposed to various TNF inhibitors revealed distinct and differential wound-healing signatures that may underlie the differential efficacy of these inhibitors in accelerating cutaneous wound healing. Taken together, these data revealed that TNF inhibitors exhibited differential levels of efficacy in accelerating cutaneous wound healing in the impaired wound-healing model in vivo.

Dose levels in particulate-containing formulations impact anti-drug antibody responses to murine monoclonal antibody in mice.

Dosage levels and particulate contents of therapeutic protein formulations are potential factors that impact immunogenicity of protein therapeutics. Here, we evaluated the effect of dose levels on the immunogenicity of protein particulates formed by adsorbing a murine monoclonal IgG2c/κ antibody (mAb1) onto silicone oil microdroplets, glass, or aluminum hydroxide (Alhydrogel) microparticles. Immune responses to these particulate-containing preparations were compared against responses to solutions of mAb1 that had been ultracentrifuged to minimize particle levels. Formulations containing 5 or 500 µg of adsorbed mAb1 were administered subcutaneously to C57BL/6J or BALB/c mice. Antidrug antibodies (ADAs) were detected using an isotype-specific enzyme-linked immunosorbent assay (ELISA) method or a chemiluminescence method. Sera from BALB/c mice showed greater ADA responses to administration of particles at the 5-µg dose level than at the 500-µg dose level. In sera from C57BL/6J mice, ADA levels detected by ELISA were independent of the particle dose levels tested. ADAs were not detected in sera from C57BL/6J mice performing the chemiluminescence technique. In conclusion, mice administered formulations of a murine antibody adsorbed onto silicone oil microdroplets, glass microparticles, or Alhydrogel(®) showed greater ADA responses that those that received particle-free mAb1 preparations, and responses were greater for formulations containing lower doses of antibody. .

A rapid and automated sample-to-result HIV load test for near-patient application.

BACKGROUND: Current viral load tests for human immunodeficiency virus (HIV) can only be performed in laboratory environments, require highly trained operators and expensive equipment, and have a turnaround time of several hours to days. The Liat HIV Quant Assay proves that such nucleic acid testing can be performed rapidly and easily, allowing application at the point of care. METHODS: The Liat Analyzer automates the entire assay, including sample preparation, amplification, and detection, in a self-contained and closed-tube system. Dynamic range, limit of detection, and subtype specificity were evaluated. Clinical samples were tested retrospectively to correlate the result of this assay with those of commercial HIV load assays. RESULTS: The Liat assay demonstrated linearity of >6 logs (R(2), 0.98) and a limit of detection of 57 copies/mL. HIV-1 group M (clades A-H), group O, and HIV-2 were detected. Testing of clinical samples showed a high degree of concordance between the copy numbers detected by the Liat assay and those detected by the Siemens and Roche assays (92.0% and 88% correlation coefficient of the log copy number, respectively). The time from sample collection to result was 88 min. CONCLUSIONS: Results suggest that the Liat HIV Quant Assay has performance equivalent to that of commercial HIV load assays and significantly reduces assay time, simplifies test operation, and provides biocontainment to allow operation in nonlaboratory settings.

Inferences about the global population structures of Cryptosporidium parvum and Cryptosporidium hominis.

Cryptosporidium parvum and Cryptosporidium hominis are two related species of apicomplexan protozoa responsible for the majority of human cases of cryptosporidiosis. In spite of their considerable public health impact, little is known about the population structures of these species. In this study, a battery of C. parvum and C. hominis isolates from seven countries was genotyped using a nine-locus DNA subtyping scheme. To assess the existence of geographical partitions, the multilocus genotype data were mined using a cluster analysis based on the nearest-neighbor principle. Within each country, the population genetic structures were explored by combining diversity statistical tests, linkage disequilibrium, and eBURST analysis. For both parasite species, a quasi-complete phylogenetic segregation was observed among the countries. Cluster analysis accurately identified recently introduced isolates. Rather than conforming to a strict paradigm of either a clonal or a panmictic population structure, data are consistent with a flexible reproductive strategy characterized by the cooccurrence of both propagation patterns. The relative contribution of each pattern appears to vary between the regions, perhaps dependent on the prevailing ecological determinants of transmission.

Genetic crosses in the apicomplexan parasite Cryptosporidium parvum define recombination parameters.

Recombinant progeny lines of Cryptosporidium parvum were generated by coinfecting immunosuppressed mice with two genetically distinct isolates of C. parvum. Progeny lines were obtained from a cross of parental lines MD x TU114 through targeted propagation in mice of progeny oocysts originating from populations lacking one parental allele at one or more loci. For each infection lineage this process was repeated until only a single allele remained for each marker, indicating that the progeny line was clonal. To study genetic recombination, 16 progeny clones were genotyped at 40 loci located on each of the eight chromosomes. The inheritance of parental alleles was significantly skewed towards the more virulent parent isolate MD. A contiguous 476 kb segment of chromosome V displayed MD allele in all progeny recovered, while MD and TU114 alleles were detected at other loci throughout the genome. The absence of alleles from one parental isolate in this chromosomal region may indicate phenotypic selection for the MD allele during the generation of these lines. A range for the meiotic crossover frequency was determined on the basis of 40 markers and the number of meioses estimated to have taken place during the crossing experiment. C. parvum exhibits a high rate of recombination commensurate with other Apicomplexa.

Cryptosporidium hominis: experimental challenge of healthy adults.

Cryptosporidium hominis causes diarrhea in humans and has been associated with community outbreaks. This study describes the infectivity, illness, and serologic response after experimental challenge of 21 healthy adult volunteers with 10-500 C. hominis (TU502) oocysts. Sixteen subjects (76.2%) had evidence of infection; the 50% infectious dose (ID(50)) was estimated to be 10-83 oocysts using clinical and microbiologic definitions of infection, respectively. Diarrhea occurred in 40% of subjects receiving 10 oocysts with a stepwise increase to 75% in those receiving 500 oocysts. A serum IgG response was seen in those receiving more than 30 oocysts. Greatest responses were seen in volunteers with diarrhea and oocyst shedding. Volunteers with no evidence of infection had indeterminant or negative IgG responses. Cryptosporidium hominis 10 oocysts) and is clinically is infectious for healthy adults (ID(50) = similar to C. parvum-induced illness. In contrast to C. parvum, C. hominis elicted a serum IgG response in most infected persons.

Emergence of distinct genotypes of Cryptosporidium parvum in structured host populations.

Cryptosporidium parvum is an apicomplexan parasite that infects humans and ruminants. C. parvum isolated from cattle in northeastern Turkey and in Israel was genotyped using multiple polymorphic genetic markers, and the two populations were compared to assess the effect of cattle husbandry on the parasite's population structure. Dairy herds in Israel are permanently confined with essentially no opportunity for direct herd-to-herd transmission, whereas in Turkey there are more opportunities for transmission as animals range over wider areas and are frequently traded. A total of 76 C. parvum isolates from 16 locations in Israel and seven farms in the Kars region in northeastern Turkey were genotyped using 16 mini- and microsatellite markers. Significantly, in both countries distinct multilocus genotypes confined to individual farms were detected. The number of genotypes per farm was higher and mixed isolates were more frequent in Turkey than in Israel. As expected from the presence of distinct multilocus genotypes in individual herds, linkage disequilibrium among loci was detected in Israel. Together, these observations show that genetically distinct populations of C. parvum can emerge within a group of hosts in a relatively short time. This may explain the frequent detection of host-specific genotypes with unknown taxonomic status in surface water and the existence of geographically restricted C. hominis genotypes in humans.

Differential evolution of repetitive sequences in Cryptosporidium parvum and Cryptosporidium hominis.

Cryptosporidium parvum and Cryptosporidium hominis are two morphologically identical species of Apicomplexan protozoa infecting humans. Although the genomes of these species are 97% identical, their host range is strikingly different. C. parvum infects humans and animals and is primarily a zoonotic infection, whereas C. hominis is typically not detected in animals. The extent of genetic polymorphism in both species has been surveyed locally, but not on a larger geographical scale. Herein, a collection of unrelated C. parvum and C. hominis isolates was genotyped using multiple, randomly distributed micro- and minisatellites. In average, minisatellites, consisting of tandemly repeated sequence motifs of 6-24 basepair, were more polymorphic than microsatellites. When the average number of micro- and minisatellite alleles per locus was used as a measure of heterogeneity, no difference between C. parvum and C. hominis was found. However, the frequency distribution of alleles in both species was significantly different and in 6 of the 14 loci the size of the C. parvum and C. hominis repeats did not overlap. Assuming that C. parvum and C. hominis evolved from a common ancestor, these observations suggest a differential evolution of repeat length at these loci.

Genotyping of Cryptosporidium parvum with microsatellite markers.

Recent outbreaks of cryptosporidiosis caused by Cryptosporidium parvum in the United States and other countries, as well as the emergence of cryptosporidiosis as a frequent cause of morbidity and mortality in immunodeficient individuals, have raised the interest of the research community in this parasite. The genus Cryptosporidium, phylum Apicomplexa, comprises an undefined number of species, of which only C. parvum is of public health concern. Cryptosporidiosis is contracted through the ingestion of oocysts, the stage of the parasite produced in large numbers by infected hosts. Because the oocysts are small, typically about 5 microm in diameter, and lack species-specific morphological features, there is a need for molecular markers to distinguish between human-infectious C. parvum and other species that do not (or only infrequently) cause disease in humans. Genetic characterization of Cryptosporidium oocysts using restriction fragment length or sequence polymorphism has revealed host-associated genotypes, that are often referred to as species. In addition, C. parvum was found to include two genotypes, designated type 1 and type 2. Type 1 is almost exclusively found in humans, whereas type 2 infects humans and various mammalian hosts. The frequent occurrence of Cryptosporidium oocysts in untreated surface water and the potential for contamination of drinking water have emphasized the need for molecular markers to track the source of oocysts within a watershed or water distribution system, and to discriminate between oocysts infectious to humans and nonpathogenic species. Genetic markers are also needed to study the taxonomy of Cryptosporidium. Several laboratories have identified microsatellites in the genome of C. parvum and have investigated the level of polymorphism at these loci. For instance, 10 alleles of marker 5B12 have been found to date among C. parvum isolates from various geographical and host origins. Multilocus haplotypes based on such markers are suitable for discriminating individual isolates of C. parvum. In an attempt to develop rapid and cost-effective methods for typing isolates of C. parvum, we have pursued two methods, a traditional polymerase chain reaction (PCR) method followed by gel electrophoresis, and real-time PCR using SYBR Green I melting curve analysis for allele identification.

Identification of genotypically mixed Cryptosporidium parvum populations in humans and calves.

Genotypic analyses of Cryptosporidium parvum oocysts have divided the species into two genotypes, referred to as type 1 and type 2. Although humans are susceptible to both types, mixed type 1/type 2 infections have rarely been identified. The paucity of mixed infections could be explained by the predominance of one type over the other in mixed infections, or by the poor sensitivity of restriction fragment length polymorphism (RFLP) analyses for detecting subpopulations. Using a type-specific real-time PCR assay capable of detecting type 1 or type 2 constituting as little as 0.01% of the population, archived and new isolates of human, bovine, and mouse origin were genotyped. Mixed type 1/type 2 infections were identified in humans and calves, including in samples previously found to be homogeneous by RFLP. Isopycnic fractionation of mixed isolates revealed that type 1 and type 2 oocysts differ in their sedimentation properties. The detection of a type 1 subpopulation in serially-propagated bovine isolates indicates that type 1 and type 2 are stably maintained during long-term passage. Together with recently reported experimental bovine and ovine type 1 infections, the persistence of type 1 subpopulation in experimentally infected animals suggests that animals may play a previously unrecognized role in the maintenance of C. parvum type 1.

Detection and genotyping of oocysts of Cryptosporidium parvum by real-time PCR and melting curve analysis.

Several real-time PCR procedures for the detection and genotyping of oocysts of Cryptosporidium parvum were evaluated. A 40-cycle amplification of a 157-bp fragment from the C. parvum beta-tubulin gene detected individual oocysts which were introduced into the reaction mixture by micromanipulation. SYBR Green I melting curve analysis was used to confirm the specificity of the method when DNA extracted from fecal samples spiked with oocysts was analyzed. Because C. parvum isolates infecting humans comprise two distinct genotypes, designated type 1 and type 2, real-time PCR methods for discriminating C. parvum genotypes were developed. The first method used the same beta-tubulin amplification primers and two fluorescently labeled antisense oligonucleotide probes spanning a 49-bp polymorphic sequence diagnostic for C. parvum type 1 and type 2. The second genotyping method used SYBR Green I fluorescence and targeted a polymorphic coding region within the GP900/poly(T) gene. Both methods discriminated between type 1 and type 2 C. parvum on the basis of melting curve analysis. To our knowledge, this is the first report describing the application of melting curve analysis for genotyping of C. parvum oocysts.