RB1 status in triple negative breast cancer cells dictates response to radiation treatment and selective therapeutic drugs.

Triple negative breast cancer (TNBC) includes basal-like and claudin-low subtypes for which only chemotherapy and radiation therapy are currently available. The retinoblastoma (RB1) tumor suppressor is frequently lost in human TNBC. Knockdown of RB1 in luminal BC cells was shown to affect response to endocrine, radiation and several antineoplastic drugs. However, the effect of RB1 status on radiation and chemo-sensitivity in TNBC cells and whether RB1 status affects response to divergent or specific treatment are unknown. Using multiple basal-like and claudin-low cell lines, we hereby demonstrate that RB-negative TNBC cell lines are highly sensitive to gamma-irradiation, and moderately more sensitive to doxorubicin and methotrexate compared to RB-positive TNBC cell lines. In contrast, RB1 status did not affect sensitivity of TNBC cells to multiple other drugs including cisplatin (CDDP), 5-fluorouracil, idarubicin, epirubicin, PRIMA-1(met), fludarabine and PD-0332991, some of which are used to treat TNBC patients. Moreover, a non-biased screen of ∼3400 compounds, including FDA-approved drugs, revealed similar sensitivity of RB-proficient and -deficient TNBC cells. Finally, ESA(+)/CD24(-/low)/CD44(+) cancer stem cells from RB-negative TNBC lines were consistently more sensitive to gamma-irradiation than RB-positive lines, whereas the effect of chemotherapy on the cancer stem cell fraction varied irrespective of RB1 expression. Our results suggest that patients carrying RB-deficient TNBCs would benefit from gamma-irradiation as well as doxorubicin and methotrexate therapy, but not necessarily from many other anti-neoplastic drugs.

High-throughput screen identifies disulfiram as a potential therapeutic for triple-negative breast cancer cells: interaction with IQ motif-containing factors.

Triple-negative breast cancer (TNBC) represents an aggressive subtype, for which radiation and chemotherapy are the only options. Here we describe the identification of disulfiram, an FDA-approved drug used to treat alcoholism, as well as the related compound thiram, as the most potent growth inhibitors following high-throughput screens of 3185 compounds against multiple TNBC cell lines. The average IC50 for disulfiram was ~300 nM. Drug affinity responsive target stability (DARTS) analysis identified IQ motif-containing factors IQGAP1 and MYH9 as direct binding targets of disulfiram. Indeed, knockdown of these factors reduced, though did not completely abolish, cell growth. Combination treatment with 4 different drugs commonly used to treat TNBC revealed that disulfiram synergizes most effectively with doxorubicin to inhibit cell growth of TNBC cells. Disulfiram and doxorubicin cooperated to induce cell death as well as cellular senescence, and targeted the ESA(+)/CD24(-/low)/CD44(+) cancer stem cell population. Our results suggest that disulfiram may be repurposed to treat TNBC in combination with doxorubicin.

Hemoglobin interactions with αB crystallin: a direct test of sensitivity to protein instability.

As a small stress response protein, human αB crystallin, detects protein destabilization that can alter structure and function to cause self assembly of fibrils or aggregates in diseases of aging. The sensitivity of αB crystallin to protein instability was evaluated using wild-type hemoglobin (HbA) and hemoglobin S (HbS), the glutamate-6-valine mutant that forms elongated, filamentous aggregates in sickling red blood cells. The progressive thermal unfolding and aggregation of HbA and HbS in solution at 37°C, 50°C and 55°C was measured as increased light scattering. UV circular dichroism (UVCD) was used to evaluate conformational changes in HbA and HbS with time at the selected temperatures. The changes in interactions between αB crystallin and HbA or HbS with temperature were analyzed using differential centrifugation and SDS PAGE at 37°C, 50°C and 55°C. After only 5 minutes at the selected temperatures, differences in the aggregation or conformation of HbA and HbS were not observed, but αB crystallin bound approximately 6% and 25% more HbS than HbA at 37°C, and 50°C respectively. The results confirmed (a) the remarkable sensitivity of αB crystallin to structural instabilities at the very earliest stages of thermal unfolding and (b) an ability to distinguish the self assembling mutant form of HbS from the wild type HbA in solution.

Short-term vitamin D receptor activation increases serum creatinine due to increased production with no effect on the glomerular filtration rate.

Vitamin D receptor activation has been associated with increased serum creatinine and reduced estimated glomerular filtration rates, raising concerns that its use may be detrimental to kidney function. Here we studied the effect of vitamin D receptor activation on serum creatinine, creatinine generation, and its clearance. We measured baseline serum creatinine and 24-h urine creatinine in 16 patients with chronic kidney disease. The measurements were repeated every day for 7 days, during which time the patients received 2 µg paricalcitol, an orally active vitamin D receptor activator, every morning. At 4 days after stopping the vitamin analog, measurements were continued for 3 days. Geometric mean parathyroid hormone levels decreased from 77 pg/ml at baseline to 43 pg/ml at the end of treatment and significantly rebounded to 87 pg/ml following paricalcitol withdrawal, thereby supporting the biological efficacy of the analog dose used. With this therapy, the serum creatinine significantly increased at a rate of 0.010 mg/dl/day and urine creatinine at a rate of 17.6 mg/day. Creatinine and iothalamate clearances did not change, whereas urine albumin decreased insignificantly. Thus, short-term vitamin D receptor activation increases creatinine generation and serum creatinine, but it does not influence the glomerular filtration rate.

Role of home blood pressure monitoring in overcoming therapeutic inertia and improving hypertension control: a systematic review and meta-analysis.

Hypertension remains the most common modifiable cardiovascular risk factor, yet hypertension control rates remain dismal. Home blood pressure (BP) monitoring has the potential to improve hypertension control. The purpose of this review was to quantify both the magnitude and mechanisms of benefit of home BP monitoring on BP reduction. Using a structured review, studies were selected if they reported either changes in BP or percentage of participants achieving a pre-established BP goal between randomized groups using home-based and office-based BP measurements. A random-effects model was used to estimate the magnitude of benefit and relative risk. The search yielded 37 randomized controlled trials with 9446 participants that contributed data for this meta-analysis. Compared with clinic-based measurements (control group), systolic BP improved with home-based BP monitoring (-2.63 mm Hg; 95% CI, -4.24, -1.02); diastolic BP also showed improvement (-1.68 mm Hg; 95% CI, -2.58, -0.79). Reductions in home BP monitoring-based therapy were greater when telemonitoring was used. Home BP monitoring led to more frequent antihypertensive medication reductions (relative risk, 2.02 [95% CI, 1.32 to 3.11]) and was associated with less therapeutic inertia defined as unchanged medication despite elevated BP (relative risk for unchanged medication, 0.82 [95% CI, 0.68 to 0.99]). Compared with clinic BP monitoring alone, home BP monitoring has the potential to overcome therapeutic inertia and lead to a small but significant reduction in systolic and diastolic BP. Hypertension control with home BP monitoring can be enhanced further when accompanied by plans to monitor and treat elevated BP such as through telemonitoring.

Serum IgG concentrations after intravenous serum transfusion in a randomized clinical trial in dairy calves with inadequate transfer of colostral immunoglobulins.

BACKGROUND: Plasma transfusions have been used clinically in the management of neonates with failure of passive transfer. No studies have evaluated the effect of IV serum transfusions on serum IgG concentrations in dairy calves with inadequate transfer of passive immunity. HYPOTHESIS: A commercially available serum product will increase serum immunoglobulin concentration in calves with inadequate transfer of colostral immunoglobulins. ANIMALS: Thirty-two Jersey and Jersey-Holstein cross calves with inadequate colostral transfer ofimmunoglobulins (serum total protein < 5.0 g/L). METHODS: Thirty-two calves were randomly assigned to either control (n = 15) or treated (n = 17) groups. Treated calves received 0.5 L of a pooled serum product IV. Serum IgG concentrations before and after serum transfusion were determined by radial immunodiffusion. RESULTS: Serum protein concentrations increased from time 0 to 72 hours in both control and transfused calves and the difference was significant between the control and treatment groups (P < .001). Mean pre- and posttreatment serum IgG concentrations in control and transfused calves did not differ significantly. Median serum IgG concentrations decreased from 0 to 72 hours by 70 mg/dL in control calves and increased over the same time interval in transfused calves by 210 mg/dL. The difference was significant between groups (P < .001). The percentage of calves that had failure of immunoglobulin transfer 72 hours after serum transfusion was 82.4%. CONCLUSIONS AND CLINICAL IMPORTANCE: Serum administration at the dosage reported did not provide adequate serum IgG concentrations in neonatal calves with inadequate transfer of colostral immunoglobulins.

Cryptosporidiosis in people: it's not just about the cows.

Cryptosporidiosis is one of the most common causes of infectious diarrhea in people. Although dairy calves are high-risk hosts, the role of other livestock, pets, and humans in the disease should not be underestimated. Some Cryptosporidium species and strains are specific to people, others are specific to animals while some are zoonotic pathogens. Cryptosporidium hominis is the species responsible for the majority of human cases in the United States, Sub-Saharan Africa, and Asia, while Cryptosporidium parvum accounts for more human cases in Europe and particularly in the United Kingdom. A deeper understanding of Cryptosporidium host range, reservoirs, and transmission is needed to develop preventive strategies to protect the general public.

Identification of survivin, an inhibitor of apoptosis, in canine urinary bladder transitional cell carcinoma.

Survivin, an inhibitor of apoptosis, is overexpressed in human invasive transitional cell carcinoma (TCC) of the urinary bladder. Survivin expression in canine TCC has not been defined. This study was designed to compare survivin expression between canine TCC and normal urinary bladder tissue. Reverse-transcriptase polymerase chain reaction (PCR) and immunohistochemistry (IHC) were performed on fresh-frozen and formalin-fixed tissues, respectively. All TCC tissues (n = 6) and 11/22 normal tissues assessed by PCR were positive for survivin. This difference was not significant (P = 0.06). With regard to IHC, 28/41 TCC samples were positive for nuclear survivin, whereas 0/46 normal tissues had nuclear immunoreactivity (P < 0.001). Cytoplasmic immunoreactivity did not significantly differ between TCC (7/41) and normal tissues (17/46) (P = 0.07). We conclude that nuclear survivin is present in canine TCC, but not in normal bladder urothelium. Future studies will evaluate the role of nuclear survivin in TCC development and as a potential therapeutic target.

Polymerase chain reaction detection of Cytauxzoon felis from field-collected ticks and sequence analysis of the small subunit and internal transcribed spacer 1 region of the ribosomal RNA gene.

Cytauxzoon felis produces a disease in domestic cats in the Midwest (U.S.A.), which often leads to a fatal outcome. Although the clinical disease process is well described, there are still many unanswered questions about this organism. For example, it is unknown whether species of ticks other than Dermacentor variabilis can serve as vectors for transmission. With recent reports of surviving cats from limited geographic areas, another relevant question is the potential for genetically less virulent organism strains. This study evaluated 352 individual or pooled tick samples (1,362 total ticks) for the presence of C. felis small subunit ribosomal RNA and internal transcribed spacer 1 (ITS-1) region genes using a polymerase chain reaction (PCR). These ticks were collected from dogs and cats in several Missouri counties, including 10 from cats diagnosed with cytauxzoonosis. Only 3 positive C. felis samples were identified in Amblyomma americanum nymphs, and there was very limited genetic variation noted in both genes. The small number of positive samples did not allow the study to determine which PCR analysis was more sensitive. This is the first known report of ITS-1 gene identification and sequencing for C. felis. It is also the first published investigation of genetic variation in C. felis.

Short communication: influence of Staphylococcus aureus intramammary infection on serum copper, zinc, and iron concentrations.

The goal of the present study was to characterize changes in serum trace mineral concentrations in cattle with experimentally induced Staphylococcus aureus mastitis. Nine primiparous Holstein-Friesian cattle were challenged with approximately 150 cfu of Staph. aureus ATCC29740 by intramammary infusion on d 6, 7, and 8 of lactation. Serum Cu, Zn, and Fe concentrations were determined immediately before and at 24, 48, and 72 h after the final intramammary infusion of Staph. aureus. Infection status (cfu/mL of Staph. aureus), milk somatic cell count, and mastitis score were also determined at these times. Infection resulted in a decrease in mean serum Cu, Zn, and Fe concentrations to 89, 83, and 81% of preinfection concentrations at 24 h postchallenge. One-way analysis of variance for repeated measures demonstrated a significant change in serum zinc concentration. The reductions in trace mineral concentrations were of less magnitude than observed following experimental E. coli mastitis.

Use of pulsed-field gel electrophoresis for detecting differences in Staphylococcus aureus strain populations between dairy herds with different cattle importation practices.

The hypothesis tested was that dairy herds which import cattle for replacement or expansion have a higher prevalence of Staphylococcus aureus mastitis and a greater number of new Staphylococcus aureus strains enter their herds than closed herds. Fifteen commercial dairy herds were divided into four groups based on cattle importation practices. Composite foremilk samples were collected at 4-monthly intervals for 1 year from all lactating cattle. Additionally, foremilk samples were collected from cattle at parturition and skin swabs were taken from the udder of primiparous heifers. All samples were cultured for Staphylococcus aureus and isolates were strain-typed using pulsed-field gel electrophoresis. Herds that purchased replacement heifers had a higher prevalence of Staphylococcus aureus mastitis than herds that purchased lactating cattle for expansion (P = 0.02). Herds that purchased replacement heifers had more total strains of Staphylococcus aureus (P = 0.01) and more new strains (P = 0.04) enter the herd than closed herds.

Influence of Staphylococcus aureus strain-type on mammary quarter milk somatic cell count and N-acetyl-beta-D-glucosaminidase activity in cattle from eight dairies.

The hypothesis tested was that there are differences in pathogenicity between strains of Staphylococcus aureus that cause bovine mastitis. Mammary quarter milk somatic cell count (SCC) and N-acetyl-beta-D-glucosaminidase (NAGase) activity were used as indicators of the pathogenicity of different strains of S. aureus that infect the bovine udder. Eight commercial dairy herds with a history of S. aureus in bulk tank milk cultures were studied. Initially, composite foremilk samples were collected from all lactating cattle in each herd and cultured for staphylococci. Subsequently, all cows with a coagulase-positive staphylococcal intramammary infection (IMI) at the initial sampling that were still present in the herd of origin had individual mammary quarter foremilk samples collected. Coagulase-positive staphylococcal isolates were confirmed as S. aureus using a commercial biotyping system. Staphylococcus aureus isolates were strain-typed using pulsed-field gel electrophoresis. Mammary quarter milk SCC and N-acetyl-beta-D-glucosaminidase activity were determined for each cow. The difference in mean somatic cell count and mean NAGase activity for mammary quarters infected with the same strain of S. aureus and for uninfected quarters on the same cow was calculated. One-way analysis of variance was used to assess differences between strains within a herd. Overall, no significant differences were found between strains, suggesting that the degree of udder parenchymal injury induced by S. aureus IMI is in general significantly affected by factors other than strain type.

The 14-3-3 cerebrospinal fluid immunoassay lacks utility in the diagnosis of clinical scrapie.

This study determined whether the immunoassay for cerebrospinal fluid 14-3-3 protein concentration was sensitive and specific in the diagnosis of naturally occurring clinical scrapie in sheep. Cerebrospinal fluid was collected from 9 sheep with the confirmed diagnosis of scrapie. Additionally, cerebrospinal fluid was collected from 13 clinically normal sheep, which originated from a closely monitored flock with no history of scrapie. Sensitivity and specificity were calculated using standard epidemiological methods. Cerebrospinal fluid immunoassay results did not differ significantly between positive and negative sheep. Test sensitivity varied from 0.55 to 0.66, depending on the choice of test endpoint. Test specificity varied from 0.30 to 0.77, depending on the choice of test endpoint. The 14-3-3 cerebrospinal fluid immunoassay appears to have no value in the diagnosis of clinical scrapie in sheep.

Association between cancer chemotherapy and canine distemper virus, canine parvovirus, and rabies virus antibody titers in tumor-bearing dogs.

OBJECTIVE: To determine the association between cancer chemotherapy and serum canine distemper virus (CDV), canine parvovirus (CPV), and rabies virus antibody titers in tumor-bearing dogs. DESIGN: Prospective study. ANIMALS: 21 client-owned dogs with various malignancies and 16 client-owned dogs with lymphoma. PROCEDURE: In study A, serum antibody titers were measured by use of hemagglutination inhibition (CPV titers) or serum neutralization (CDV titers) before and at least 1 month after initiation of chemotherapy. Baseline values were compared with values obtained from a control population of 122 healthy dogs seen for routine revaccination. Titers were considered protective at > or = 1:96 for CDV and > or = 1:80 for CPV. In study B, serum IgG titers were measured by use of immunofluorescent assay (CDV and CPV titers) and rapid fluorescent focus inhibition test (RFFIT, rabies titers) at baseline and again at weeks 5, 8, and 24 of a standard chemotherapy protocol for treatment of lymphoma. An IgG titer of > or = 1:50 was considered protective for CPV and CDV. An RFFIT titer of > or = 0.5 U/ml was considered protective for rabies virus. RESULTS: Significant changes were not detected in CDV, CPV, and rabies virus titers following chemotherapy in tumor-bearing dogs. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that established immunity to CDV, CPV, and rabies virus from previous vaccination is not significantly compromised by standard chemotherapy used to treat tumor-bearing dogs.

Transferable residues from dog fur and plasma cholinesterase inhibition in dogs treated with a flea control dip containing chlorpyrifos.

We studied chlorpyrifos, an insecticide present in a commercial dip for treating ectoparasites in dogs, to estimate the amount of transferable residues that children could obtain from their treated pets. Although the chlorpyrifos dip is no longer supported by the manufacturer, the methodology described herein can help determine transferable residues from other flea control insecticide formulations. Twelve dogs of different breeds and weights were dipped using the recommended guidelines with a commercial, nonprescription chlorpyrifos flea dip for 4 consecutive treatments at 3-week intervals (nonshampoo protocol) and another 12 dogs were dipped with shampooing between dips (shampoo protocol). The samples collected at 4 hr and 7, 14, and 21 days after treatment in the nonshampoo protocol averaged 971, 157, 70, and 26 microg chlorpyrifos, respectively; in the shampoo protocol the samples averaged 459, 49, 15, and 10 microg, respectively. The highest single sample was about 7,000 microg collected at 4 hr. The pretreatment specific activities in the plasma of the dogs were about 75 nmol/min/mg protein for butyrylcholinesterase (BChE), and 9 nmol/min/mg protein for acetylcholinesterase (AChE). BChE was inhibited 50-75% throughout the study, and AChE was inhibited 11-18% in the nonshampoo protocol; inhibition was not as great in the shampoo protocol. There was no correlation (p

Serum immunoglobulin G concentrations in calves fed fresh and frozen colostrum.

OBJECTIVE: To determine whether serum IgG concentrations in neonatal calves are adversely affected by short-term frozen storage of colostrum. DESIGN: Prospective study. SAMPLE POPULATION: Experiment 1 consisted of 10 pairs of Holstein calves (n = 20) fed matched aliquots of either fresh (n = 10) or frozen and thawed (10) colostrum. In experiment 2, 26 Holstein calves were fed either fresh (n = 13) or frozen and thawed (n = 13) colostrum. PROCEDURE: Experiment 1 consisted of calves resulting from observed parturitions; calves were randomly assigned to treatment groups (fresh or frozen and thawed colostrum) in pairs. Calves were fed 4 L aliquots of colostrum via oroesophageal intubation at 3 hours of age. Serum IgG concentrations at 2 days of age were compared between the 2 groups by use of a paired t-test. Experiment 2 consisted of calves resulting from observed parturitions; calves were randomly assigned to treatment groups (fresh or frozen and thawed colostrum). Calves were fed 4 L aliquots of colostrum via oroesophageal intubation at 3 hours of age. Regression analysis was used to determine whether calf serum IgG concentration was a function of colostral IgG concentration and colostrum storage group. RESULTS: Significant differences were not observed between the 2 groups in experiment 1. No significant relationship was observed between colostrum storage group and serum IgG concentration in experiment 2. The model that best predicted serum IgG concentrations accounted for 20% of the variability in serum IgG concentration. CONCLUSION AND CLINICAL RELEVANCE: Frozen colostrum is an adequate source of IgG for calves.

Clinical assessment of a chemosensitivity assay as a treatment planning tool for dogs with cancer.

This study evaluated the clinical utility of a commercially available chemosensitivity assay. In the first part of the study, tumor tissues from dogs with various malignancies were tested, and the dogs were treated with a mitoxantrone/cyclophosphamide combination protocol. Tumor response was evaluated and compared to the predicted response. Assay results were not a significant predictor of clinical response to chemotherapy or of survival time. In the second part of the study, assay results were used to direct therapy in dogs with refractory lymphoma. There was no significant correlation (p equals 0.323) between predicted response and case outcome.