Phenylbutazone induces equine glandular gastric disease without decreasing prostaglandin E2 concentrations.

In equids, phenylbutazone at high doses induces gastric disease, primarily in the glandular portion of the stomach. However, the mechanism of nonsteroidal anti-inflammatory drug (NSAID)-induced gastric disease in horses has yet to be determined. While phenylbutazone-associated ulceration is often attributed to a decrease in basal gastric prostaglandins, this has not been demonstrated in the horse. Twelve horses were randomly assigned to treatment (n = 6; 4.4 mg/kg phenylbutazone PO in 20 ml molasses q 12 hr for 7 days) or placebo (n = 6; 20 ml molasses PO q 12 hr for 7 days) groups. Before treatment and 3 and 7 days after initiation of treatment, gastroscopy was performed and glandular gastric biopsies were collected and frozen at -80°C. Glandular disease was assessed on a scale of 0-4. Prostaglandin E2 concentrations in biopsies were measured using a commercially available enzyme-linked immunosorbent assay. All phenylbutazone-treated horses developed grade ≥2 glandular disease. Prostaglandin concentrations increased over time (p = .0017), but there was no effect of treatment (p = .49). These findings indicate that despite induction of glandular disease grade ≥2, phenylbutazone did not decrease basal glandular gastric prostaglandin E2 concentration.

Monoclonal antibody capture and viral clearance by cation exchange chromatography.

Traditionally, post-production culture harvest capture of therapeutic monoclonal antibodies (mAbs) is performed using Protein A chromatography. We investigated the efficiency and robustness of cation exchange chromatography (CEX) in an effort to evaluate alternative capture methodologies. Up to five commercially available CEX resins were systematically evaluated using an experimentally optimized buffer platform and a design-of-experiment (DoE) approach for their ability to (a) capture a model mAb with a neutral isoelectric point, (b) clear three model viruses (porcine parvovirus, CHO type-C particles, and a bacteriophage). This approach identified a narrow operating space where yield, purity, and viral clearance were optimal under a CEX capture platform, and revealed trends between viral clearance of PPV and product purity (but not yield). Our results suggest that after unit operation optimization, CEX can serve as a suitable capture step.

Lymphopenia and interleukin-2 therapy alter homeostasis of CD4+CD25+ regulatory T cells.

CD4(+)CD25(+) regulatory T (T(reg)) cells have a crucial role in maintaining immune tolerance. Mice and humans born lacking T(reg) cells develop severe autoimmune disease, and depletion of T(reg) cells in lymphopenic mice induces autoimmunity. Interleukin (IL)-2 signaling is required for thymic development, peripheral expansion and suppressive activity of T(reg) cells. Animals lacking IL-2 die of autoimmunity, which is prevented by administration of IL-2-responsive T(reg) cells. In light of the emerging evidence that one of the primary physiologic roles of IL-2 is to generate and maintain T(reg) cells, the question arises as to the effects of IL-2 therapy on them. We monitored T(reg) cells during immune reconstitution in individuals with cancer who did or did not receive IL-2 therapy. CD4(+)CD25(hi) cells underwent homeostatic peripheral expansion during immune reconstitution, and in lymphopenic individuals receiving IL-2, the T(reg) cell compartment was markedly increased. Mouse studies showed that IL-2 therapy induced expansion of existent T(reg) cells in normal hosts, and IL-2-induced T(reg) cell expansion was further augmented by lymphopenia. On a per-cell basis, T(reg) cells generated by IL-2 therapy expressed similar levels of FOXP3 and had similar potency for suppression compared to T(reg) cells present in normal hosts. These studies suggest that IL-2 and lymphopenia are primary modulators of CD4(+)CD25(+) T(reg) cell homeostasis.

Women having abortion in urban Nepal: 2005 and 2010 compared.

BACKGROUND: The use of abortion services at the Maternity Hospital clinic, the largest public sector abortion clinic in Nepal, has risen over the years. Whether the profile of the clients, reasons for abortion, and contraceptive use have changed are not known and need to be investigated. OBJECTIVES: This paper evaluates changes between 2005 and 2010 in the socio-demographic profile of abortion users, reasons for seeking abortion, and contraceptive use of two cohorts of women who had first-trimester abortion at the Maternity Hospital. METHODS: We used data from two similar surveys conducted in 2005 and 2010 among 672 and 392 women, respectively, who obtained first-trimester surgical abortion in a large public sector clinic. We analyzed trend data in service utilization and carried out a cost analysis. RESULTS: The number of women having abortions has steadily increased over the years, and cumulatively about 19,800 women have received services. The profile of the clients at this clinic remained essentially the same between 2005 and 2010. The typical users of abortion services at the clinic has were 27 years old with two living children, mostly married, with the majority not wanting to have more children. About half of them used a contraceptive method-mostly condoms, withdrawal, the pill and rhythm-in the month of unintended pregnancy, suggesting failures with these methods. Health concerns, dislike of available methods, and perceived low risk of pregnancy were common reasons for not using a contraceptive method. CONCLUSION: Despite increases in the number of clients, the socio-demographic profile of the abortion clients has remained similar over the years. The linkage between the abortion and family planning clinics needs to be strengthened.

Mechanistic insights into the formation of polyion complex aggregates from cationic thermoresponsive diblock copolymers.

HYPOTHESIS: The formation of polyion complexes (PICs) comprising thermoresponsive polymers is intended to result in the formation of aggregates that undergo significant structural changes with temperature. Moreover the observed modifications might be critically affected by polymer structure and PICs composition. EXPERIMENTS: Different block copolymers based on cationic poly(3-acrylamidopropyltrimethylammonium chloride) and thermoresponsive poly(N-isopropylacrylamide) were synthesized by aqueous RAFT/MADIX polymerization at room temperature. Addition of poly(acrylic acid) in a controlled fashion led to the formation of PICs aggregates. The structural changes induced by temperature were characterized by differential scanning calorimetry, Nuclear Magnetic Resonance spectroscopy and scattering methods. FINDINGS: Thermoresponsive PICs undergo significant structural changes when increasing temperature above the cloud point of the thermoresponsive block. The reversibility of these phenomena depends strongly on the structural parameters of the block copolymers and on PICs composition.

Process analytical technology (PAT) for biopharmaceutical products: Part I. concepts and applications.

Process analytical technology (PAT) has been gaining momentum in the biotech community due to the potential for continuous real-time quality assurance resulting in improved operational control and compliance. In this two part series, we address PAT as it applies to processes that produce biotech therapeutic products. In the first part, we address evolution of the underlying concepts and applications in biopharmaceutical manufacturing. We also present a literature review of applications in the areas of upstream and downstream processing to illustrate how implementation of PAT can help realize advanced approaches to ensuring product quality in real time. In the second part, we will explore similar applications in the areas of drug product manufacturing, rapid microbiology, and chemometrics as well as evolution of PAT in biotech processing.

Process analytical technology (PAT) for biopharmaceutical products: Part II. Concepts and applications.

Implementing real-time product quality control meets one or both of the key goals outlined in FDA's PAT guidance: "variability is managed by the process" and "product quality attributes can be accurately and reliably predicted over the design space established for materials used, process parameters, manufacturing, environmental, and other conditions." The first part of the paper presented an overview of PAT concepts and applications in the areas of upstream and downstream processing. In this second part, we present principles and case studies to illustrate implementation of PAT for drug product manufacturing, rapid microbiology, and chemometrics. We further present our thoughts on how PAT will be applied to biotech processes going forward. The role of PAT as an enabling component of the Quality by Design framework is highlighted. Integration of PAT with the principles stated in the ICH Q8, Q9, and Q10 guidance documents is also discussed.

Detection, isolation, and continuous production of cytopathic retroviruses (HTLV-III) from patients with AIDS and pre-AIDS.

A cell system was developed for the reproducible detection of human T-lymphotropic retroviruses (HTLV family) from patients with the acquired immunodeficiency syndrome (AIDS) or with signs or symptoms that frequently precede AIDS (pre-AIDS). The cells are specific clones from a permissive human neoplastic T-cell line. Some of the clones permanently grow and continuously produce large amounts of virus after infection with cytopathic (HTLV-III) variants of these viruses. One cytopathic effect of HTLV-III in this system is the arrangement of multiple nuclei in a characteristic ring formation in giant cells of the infected T-cell population. These structures can be used as an indicator to detect HTLV-III in clinical specimens. This system opens the way to the routine detection of HTLV-III and related cytopathic variants of HTLV in patients with AIDS or pre-AIDS and in healthy carriers, and it provides large amounts of virus for detailed molecular and immunological analyses.

Multicenter study on in vitro characterization of dendritic cells.

BACKGROUND: There is growing interest in the use of in vitro-expanded dendritic cells (DC) in cancer immunotherapy as cellular-based vaccines. However, the methods used for in vitro preparation vary widely between institutions. Therefore, a strong need exists for standardization, characterization and quality control (QC) of such vaccines. A first prospective multicenter pilot study was performed to investigate basic QC parameters of frozen/thawed DC. The study design was focused on comparison of test results for cell counts, immunophenotyping and cell viability. METHODS: CD14+ monocytes were isolated from three healthy volunteers. The cells were expanded in vitro, matured and cryopreserved using a standardized protocol in one laboratory. The aliquots of cryopreserved DC and a panel of reagents were shipped to eight laboratories worldwide. The objective was to compare the results of non-functional QC assays between sites by testing identical DC vaccines and using a pre-defined test protocol. RESULTS: Measurements of nucleated cell (NC) content of thawed DC vaccines with different types of hematology analyzers (HA) gave similar results for the majority of sites. Immunophenotyping using identical clones of monoclonal antibodies for the detection of surface antigens (i.e. CD1a, CD14, CD16, CD83, CD86 and HLA-DR) provided mostly comparable results between laboratories with an acceptable level of variation. In contrast, highly different results between study sites were generated for measuring the viability of thawed DC by flow cytometry using 7-amino-actinomycin D (7-AAD) dye exclusion. DISCUSSION: In characterizing frozen/thawed DC vaccines, NC counts generated by HA yielded similar results between different laboratories. Furthermore, immunophenotyping of DC vaccines can be standardized between centers, i.e. by using identical reagents. Because of highly variable results between laboratories, 7-AAD viability testing of thawed DC needs to be studied further to identify potential causes for the observed variability.

A multicenter comparison study between the Endosafe PTS rapid-release testing system and traditional methods for detecting endotoxin in cell-therapy products.

BACKGROUND: Rapid-release testing reduces the waiting period for administration of time-sensitive cell-therapy products. Current assay systems are labor intensive and time consuming. The Endosafe portable test system (PTS) is a chromogenic Limulus amebocyte lysate (LAL) portable endotoxin detection system that provides quantitative results in approximately 15 min. To evaluate Endosafe performance with cell-therapy products, side-by-side testing of traditional LAL systems and the Endosafe system was conducted at the Production Assistance for Cellular Therapies (PACT) facilities and the National Institutes of Health's Department of Transfusion Medicine, USA. METHODS: Charles River Laboratories provided each center with a PTS reader and two commercially prepared lyophilized reference standard endotoxin (RSE) vials. All samples tested with the Endosafe system used 0.05-5.0 endotoxin unit/mL (EU/mL) sensitivity cartridges provided by Charles River. Each vial was reconstituted with LAL water and tested in triplicate using the Endosafe and in-house LAL methods. Subsequently, each center tested the endotoxin content of standard dilutions of cell-therapy products, thus creating paired test results for each sample. Additionally, fabricated endotoxin-positive samples containing varying concentrations of endotoxin were prepared and shipped to all centers to perform blinded testing. RESULTS: Valid paired results, based on each center's LAL method and the Endosafe system criteria, were analyzed. Endotoxin detection between paired results was equivalent in most cases. DISCUSSION: The Endosafe system provided reliable results with products typically produced in cell-therapy manufacturing facilities, and would be an appropriate test on which to base the release of time-sensitive cell-therapy products.

Risk factors for equine glandular and squamous gastric disease in show jumping Warmbloods.

BACKGROUND: Prevalence of, and risk factors for, equine squamous gastric disease (ESGD) are well established. Limited data exists on risk factors for equine glandular gastric disease (EGGD). OBJECTIVES: To identify management factors associated with EGGD in show jumping Warmbloods in training. A secondary objective was to identify management factors associated with ESGD. STUDY DESIGN: Cross-sectional. METHODS: Gastroscopies were performed in horses following a 12-16 h fast. Management questionnaires were collected for each horse. Risk factors were determined using multivariable logistic regression modelling. RESULTS: Eighty-three horses were included in the final analysis. Exercising ≥6 days per week increased the odds of EGGD grade ≥1/4 (odds ratio [OR] = 3.5; 95% confidence interval [CI] 1.2-10.7) compared to less frequent exercise. Currently showing increased the risk of EGGD grade ≥2/4 (OR = 10.2; 95% CI, 1.04-100), while competing at the international level decreased the odds of EGGD grade ≥2/4 (OR = 0.11; 95% CI, 0.01-0.97). Exercise intensity increased the odds of grade ≥1/4 ESGD (OR = 2.8; 95% CI, 1.03-7.8) and feeding beet pulp decreased odds (OR = 0.22; 95% CI, 0.07-0.7). Exercise intensity (OR = 3.8; 95% CI, 1.1-12.8) increased the likelihood of grade ≥2/4 ESGD and feeding beet pulp decreased the odds of grade ≥2/4 ESGD (OR = 0.1; 0.02-0.64) respectively. MAIN LIMITATIONS: This study used a convenience sample of horses within a relatively small (approximately 200 km) geographic radius. The sample size was relatively small, particularly within the international competition level group. CONCLUSIONS: Training and feeding strategies and competition level appear to influence the occurrence of EGGD and ESGD. Prospective studies evaluating the impact of training frequency, duration, and intensity on gastric physiology may clarify the role of exercise in gastric disease.

Actin- and tubulin-dependent functions during Saccharomyces cerevisiae mating projection formation.

Several conditional-lethal mutant alleles of the single-copy Saccharomyces cerevisiae beta-tubulin and actin genes were used to evaluate the roles of microtubules and actin filaments in the pheromone-induced extension of mating projections. Mutants defective in tubulin assembly form projections indistinguishable in appearance from those formed by wild-type cells. However, the tubulin mutants are unable to move their nuclei into the projections and to orient the spindle pole body associated with each nucleus toward the projection tip. Actin mutants are defective in spatial orientation of cell-surface growth required for formation of normal mating projections. Migration of nuclei into mating projections and Spa2p segregation to projection tips are also defective in actin mutants. Studies with abp1 null mutants showed that the function of the Abp1p actin-binding protein is either not required for projection formation or there are other proteins in yeast with similar functions. Our findings demonstrate that actin is required to restrict cell-surface growth to a defined region for pheromone-induced morphogenesis and suggest that nuclear position and orientation in mating projections depend on direct or indirect interaction of microtubules with actin filaments.

In vivo distribution of adoptively transferred indium-111-labeled tumor infiltrating lymphocytes and peripheral blood lymphocytes in patients with metastatic melanoma.

Patients with metastatic melanoma undergoing therapy with cyclophosphamide (CPM), tumor-infiltrating lymphocytes (TIL), and interleukin-2 (IL-2) were studied for the ability of their 111In-labeled TIL or peripheral blood lymphocytes (PBL) to localize in sites of tumor using gamma camera imaging and biopsies. Nineteen infusions of radiolabeled TIL were given to 18 patients, while five patients received radiolabeled autologous PBL during TIL therapy. Clear tumor localization was seen on 13 of 18 nuclear scan series performed on 111In-TIL recipients, while tumor was imaged in only one of four scan sequences on patients given 111In-PBL. Nineteen paired biopsies of tumor and normal skin were completed on 10 patients receiving 111In-TIL, while eight biopsies were done on three PBL patients receiving 111In-PBL. The mean percentage of total injectate activity localizing per gram of tumor tissue was 0.0049% in the TIL group and 0.0010% in the PBL group (P2 = .0004). The mean of the tumor to normal skin ratios of the 111In-TIL group was three times that for 111In-PBL (P2 = .0072). One patient was studied by nuclear scanning on three consecutive treatment courses of CPM, TIL, and IL-2. He initially demonstrated clear tumor localization by 111In-TIL at several sites, then faint localization with 111In-PBL at a single site, and subsequently positive tumor imaging on repeat 111In-TIL infusion at multiple sites. These results confirm and expand our initial data demonstrating that human TIL transferred with CPM pretreatment and followed by IL-2 preferentially localize to tumor sites and indicate that this localization is greater for TIL than PBL.

Enhancing activity of various immunoaugmenting agents on the delayed-type hypersensitivity response in mice.

Six immunoaugmenting agents were tested in the delayed-type hypersensitivity reaction (DTH) in normal BALB/c X DBA/2 mice. The agents tested, levan, lentinan, mannozym, maleic anhydride divinyl ether, polyriboinosinic-polycytidylic acid-poly-L-lysine, and highly purified L-cell interferon, gave significant increases in the DTH response above the sheep red blood cell control. The schedule of doses for each agent corresponded with previous experiments from this laboratory of the maximum natural killer cell activity, macrophage activation, and interferon induction. Highly purified L-cell interferon was capable of eliciting a significant DTH response when given 4 hr after the initial challenge with sheep erythrocytes. In addition, lambda-carrageenan, a macrophage-cytotoxic agent which can render the macrophage inactive, was found to suppress the DTH response to levels slightly above phosphate-buffered saline controls. The carrageenan-induced suppression of the DTH response could be abrogated by coadministration with immunoaugmenting agents to levels attained with the immunoaugmenting agents alone.

Tumor localization of adoptively transferred indium-111 labeled tumor infiltrating lymphocytes in patients with metastatic melanoma.

Lymphoid cells infiltrating into human tumors can be expanded in vitro in medium containing interleukin-2 (IL-2). Adoptive transfer of these tumor-infiltrating lymphocytes (TIL) mediates potent antitumor effects in murine tumor models. Clinical trials to evaluate the efficacy of these cells in patients with advanced cancer are underway. We have investigated whether infused TIL labeled with indium 111 (111In) oxine can traffic and localize to metastatic deposits of tumor. Six patients with metastatic malignant melanoma who had multiple sites of subcutaneous, nodal, and/or visceral disease were the subjects of the study. The patients received cyclophosphamide 36 hours before receiving the intravenous (IV) infusion of TIL followed by IL-2 IV every eight hours. The distribution and localization of the TIL were evaluated using serial whole body gamma camera imaging, serial blood and urine samplings, and serial biopsies of tumor and normal tissue. 111In-labeled TIL localized to lung, liver, and spleen within two hours after the infusion of activity. Activity in the lung diminished within 24 hours. As early as 24 hours after injection of 111In-labeled TIL, localization of TIL to sites of metastatic deposits was demonstrated in all six patients using either imaging studies or biopsy specimens or both. 111In activity in tumor tissue biopsies ranged from three to 40 times greater than activity in normal tissue. A progressive increase in the radioactive counts at sites of tumor deposit was seen. This study shows that labeled TIL can localize preferentially to tumor, and provides information concerning the possible mechanism of the therapeutic effects of TIL.

Molecular evolution of the telomere-associated MAL loci of Saccharomyces.

The MAL gene family of Saccharomyces consists of five multigene complexes (MAL1, MAL2, MAL3, MAL4, and MAL6) each of which encodes maltose permease (GENE 1), maltase (GENE 2) and the trans-acting MAL-activator (GENE 3). Four of these loci have been mapped and each is located at or near the telomere of a different chromosome. We compare the physical structure of the MAL loci and their flanking sequences. The MAL loci were shown to be both structurally and functionally homologous throughout an approximately 9.0-kb region. The orientation of the MAL loci was determined to be: CENTROMERE . . . GENE 3-GENE 1-GENE 2 . . . TELOMERE. Telomere-adjacent sequences were found flanking GENE 2 of the MAL1, MAL3 and MAL6 loci. No common repeated elements were found on the centromere-proximal side of all the MAL1, loci. These results suggest that, during the evolution of this polygenic family, the MAL loci translocated to different chromosomes via a mechanism that involved the rearrangement(s) of chromosome termini.

GATA factors and the origins of adult and embryonic blood in Xenopus: responses to retinoic acid.

The transcription factors, GATA-1, -2 and -3 play essential roles in the differentiation of haematopoietic cells. To study the process of blood formation during vertebrate development we have used the expression of these GATA factors to locate haematopoietic cells in Xenopus embryos and to act as sensors for the effects of all-trans retinoic acid (RA), a signalling molecule which influences both anteroposterior patterning and haematopoietic differentiation. GATA factor expression was detected in the leading edge of the gastrulating mesoderm, in the ventral blood island (VBI) and dorsolateral plate (DLP) mesoderms and in a population of cells between the VBI and DLP. The VBI contributes to both embryonic and adult blood, whereas the DLP contains precursors of adult blood only, which have not been identified previously with molecular markers. The possibility that the GATA-2-expressing cells between the VBI and DLP were haematopoietic progenitors migrating from the VBI to the DLP was ruled out by transplantation analysis. Differential effects of RA on the expression of GATA-1 and GATA-2 suggest that RA has a direct action on haematopoietic differentiation, rather than on the formation of haematopoietic mesoderm.

Paclitaxel chemotherapy after autologous stem-cell transplantation and engraftment of hematopoietic cells transduced with a retrovirus containing the multidrug resistance complementary DNA (MDR1) in metastatic breast cancer patients.

The MDR1 multidrug resistance gene confers resistance to natural-product anticancer drugs including paclitaxel. We conducted a clinical gene therapy study to determine whether retroviral-mediated transfer of MDR1 in human hematopoietic cells would result in stable engraftment, and possibly expansion, of cells containing this gene after treatment with myelosuppressive doses of paclitaxel. Patients with metastatic breast cancer who achieved a complete or partial remission after standard chemotherapy were eligible for the study. Hematopoietic stem cells (HSCs) were collected by both peripheral blood apheresis and bone marrow harvest after mobilization with a single dose of cyclophosphamide (4 g/m2) and daily filgrastim therapy (10 microg/kg/day). After enrichment for CD34+ cells, one-third of each collection was incubated ex vivo for 72 h with a replication-incompetent retrovirus containing the MDR1 gene (G1MD) in the presence of stem-cell factor, interleukin 3, and interleukin 6. The remaining CD34+ cells were stored without further manipulation. All of the CD34+ cells were reinfused for hematopoietic rescue after conditioning chemotherapy with ifosfamide, carboplatin, and etoposide regimen. After hematopoietic recovery, patients received six cycles of paclitaxel (175 mg/m2 every 3 weeks). Bone marrow and serial peripheral blood samples were obtained and tested for the presence of the MDR1 transgene using a PCR assay. Six patients were enrolled in the study and four patients received infusion of genetically altered cells. The ex vivo transduction efficiency, estimated by the PCR assay, ranged from 0.1 to 0.5%. Three of the four patients demonstrated engraftment of cells containing the MDR1 transgene. The estimated percentage of granulocytes containing the MDR1 transgene ranged from a maximum of 9% of circulating nucleated cells down to the limit of detection of 0.01%. One patient remained positive for the MDR1 transgene throughout all six cycles of paclitaxel therapy, whereas the other 2 patients showed a decrease in the number of cells containing the transgene to undetectable levels. Despite the low level of engraftment of MDR1-marked cells, a correlation was observed between the relative number of granulocytes containing the MDR1 transgene and the granulocyte nadir after paclitaxel therapy. No adverse reactions to the genetic manipulation procedures were detected. Therefore, engraftment of human HSCs transduced with the MDR1 gene can be achieved. However, the overall transduction efficiency and stable engraftment of gene-modified HSCs must be improved before MDR1 gene therapy and in vivo selection with anticancer drugs can be reliably used to protect cancer patients from drug-related myelosuppression.