Search

During a 6-mo period, two 5-6 mo old female chinchillas (Chinchilla lanigera) were examined at the University of Colorado Anschutz Medical Campus after the discovery of firm, nonmobile masses in the left ventral cervical and left axillary region. Other than these findings and mild weight loss, both chinchillas' physical exams were normal. Bloodwork revealed an inflammatory leukogram characterized by leukocytosis, toxic neutrophils, lymphopenia, and monocytosis with mild, nonregenerative anemia. At necropsy, both masses were identified as abscesses. Streptococcus equi, subspecies zooepidemicus (S. zooepidemicus) was isolated in pure culture. Histology of the lungs, liver, spleen, and kidneys showed a marked increase in the numbers of both polymorphonuclear leukocytes and lymphocytes. Both animals were deemed unsuitable for research and were euthanized under isoflurane anesthesia by an intracardiac injection of pentobarbital sodium solution. S. zooepidemicus is an opportunistic, commensal organism found in the upper respiratory tract of horses. This organism has been documented to cause disease in other species and is zoonotic. Infections in humans have been reported, resulting in glomerulonephritis, endocarditis, septic arthritis, osteomyelitis, meningitis, and death. To aid in diagnosis and prospective surveillance of this bacteria, oral and nasal swabs were collected from the remaining cohort of chinchillas, and a qPCR screening assay was implemented. Within 12 mo, 4 of 41 additional females tested positive by culture or qPCR, resulting in a disease prevalence of 14% (6 of 43). However, only 2 of the additional 4 S. zooepidemicus positive animals developed clinical signs. The potential for the spread of infection, zoonosis, and adverse effects on research demonstrate that surveillance for S. zooepidemicus should be considered in a biomedical research environment.

Purpose This pilot study investigated the in vivo therapeutic potential and tolerability of a multimodal ophthalmic formulation, topical eye drops (TED), for acute mustard gas keratopathy (MGK) using a rabbit model. Methods Twenty New Zealand White rabbits were used. Only right eyes of 18 rabbits (oculus dexter [OD]) received single sulfur mustard gas (SM) vapor injury, whereas contralateral eyes were left untreated or received TED for tolerabilty evaluation. Two rabbit eyes received no treatment and served as age-matched naive control. The four groups were: Naive (oculus sinister [OS] untreated eyes; n = 9); TED (OS treated only with TED BID for 3 days; n = 9); SM (OD exposed to SM vapor; n = 9); and SM+TED (OD exposed to SM+TED BID for 3 days; n = 9). Ocular examination in live rabbits were performed utilizing slit-lamp biomicroscopy, Fantes grading system, fluorescein staining, Schirmer's tests, pachymetry, and applanation tonometry. Cellular and molecular changes in rabbit corneas were assessed after humane euthanasia on day-3 and day-7 with histopathological and real-time polymerase chain reaction PCR techniques. Results TED to rabbit eyes was found tolerable in vivo. SM-exposed eyes showed significant increase in Fantes scores, central corneal thickness (CCT), Schirmer's test, epithelium-stroma separation, and corneal edema. TED mitigated clinical symptoms by reducing corneal edema, Fantes scores, CCT, and Schirmer's test. Further, TED decreased SM-induced corneal haze, inflammatory and profibrotic markers, transforming growth factor-TGF-β1 and cyclooxygenase-2COX-2, and damage to corneal structure, including epithelial-stromal integrity. Conclusions The developed multimodal eyedrop formulation, TED, has potential to mitigate acute MGK effectively in vivo. Translational relevance TED is effective against MGK.

Acrolein is a highly reactive and volatile unsaturated aldehyde commonly used for producing scores of commercial products. It has been recognized as a chemical weapon since its use during World War I, and more recently, in Syria. Acrolein exposure causes severe eye, skin, and lung damage in addition to many casualties. In the eye, it causes severe pain, eyelid swelling, corneal burns, and vision impairment. Very little information is available about how acrolein damages the cornea and causes vision loss. At present, the lack of clinically relevant animal models limits evaluation of acrolein toxicity and mechanisms specific to the eye. We aim to standardize the mode of delivery and exposure duration of acrolein, damaging the rabbit eye in vivo as an ocular injury model for studying the toxicity of acrolein and developing medical countermeasures. Rabbit eyes were exposed to two modes of delivery (topical and vapor) for different durations (1-5 minutes). Clinical ophthalmic examinations with a slit lamp, stereomicroscope, fluorescein dye, pachymeter, tonometer, and tearing examinations in live rabbits were performed at various times up to 4 weeks. Corneas were histologically diagnosed for transparency, fibrosis, collagens, and neovascularization. Our study successfully established an in vivo rabbit model for evaluating acrolein toxicity to the eye, accounting for different modes and durations of exposure.

Wound healing differs significantly between men and women in a tissue-dependent manner. Dermal wounds heal faster in women whereas mucosal wounds heal faster in men. However, the effect of sex as a variable in corneal wound healing is largely unknown. The primary objective of this study was to test whether sex is a biological variable in corneal wound healing activated by the trauma or injury using an established in vivo rabbit model with male and female New Zealand White rabbits. Corneal wounds in rabbits were produced by a single topical alkali (0.5N Sodium hydroxide) application. Serial slit-lamp, stereo biomicroscopy, and applanation tonometry evaluated corneal opacity, anterior segment ocular health, and intraocular pressure (IOP), respectively, at various times during the study. Fourteen days after alkali-wound, corneal tissues were collected after humane euthanasia to examine cellular and molecular wound healing parameters. Quantitative PCR (qPCR) and immunofluorescence were used to quantify changes in the extracellular modeling protein levels of alpha-smooth muscle actin (α-SMA), Fibronectin (FN), Collagen-I (Col-I), and Transforming growth factor beta 1 (TGFβ1) involved in corneal healing. Hematoxylin and Eosin (H&E) staining was used to study histopathological changes in morphology and TUNEL assay to evaluate levels of apoptotic cell death. Male and female rabbits showed no significant differences in corneal opacity (Fantes score) or intraocular pressure (IOP) values (9.5 ± 0.5 mm Hg) in live animals. Likewise, no statistically significant sex-based differences in the mRNA levels of α-SMA (male = 5.95 ± 0.21 fold vs. female = 5.32 ± 0.043), FN (male = 3.02 ± 0.24 fold vs. female = 3.23 ± 0.27), Col-I (male = 3.12 ± 0.37 fold vs. female = 3.31 ± 0.24), TGFβ1 (male = 1.65 ± 0.06 fold vs. female = 1.59 ± 0.053); and protein levels of α-SMA (male = 74.16 ± 4.6 vs. female = 71.58 ± 7.1), FN (male = 60.11 ± 4.6 vs. female = 57.41 ± 8.3), Col-I (male = 84.11 ± 2.8 vs. female = 84.55 ± 3.6), TGFβ1 (male = 11.61 ± 2.8 vs. female = 9.5 ± 3.04) were observed. Furthermore, H&E and TUNEL analyses found no statistically significant differences in cellular structures and apoptosis, respectively, in male vs. female corneas. Consistent with earlier reports, wounded corneas showed significantly increased levels of these parameters compared to the unwounded corneas. Our data suggest that sex is not a major biological variable during active early stages of corneal wound healing in rabbits in vivo.

Vision impairment from corneal fibrosis is a common consequence of irregular corneal wound healing after injury. Intermediate-conductance calmodulin/calcium-activated K+ channels 3.1 (KCa3.1) play an important role in cell cycle progression and cellular proliferation. Proliferation and differentiation of corneal fibroblasts to myofibroblasts can lead to corneal fibrosis after injury. KCa3.1 has been shown in many non-ocular tissues to promote fibrosis, but its role in corneal fibrosis is still unknown. In this study, we characterized the expression KCa3.1 in the human cornea and its role in corneal wound healing in vivo using a KCa3.1 knockout (KCa3.1-/-) mouse model. Additionally, we tested the hypothesis that blockade of KCa3.1 by a selective KCa3.1 inhibitor, TRAM-34, could augment a novel interventional approach for controlling corneal fibrosis in our established in vitro model of corneal fibrosis. The expression of KCa3.1 gene and protein was analyzed in human and murine corneas. Primary human corneal fibroblast (HCF) cultures were used to examine the potential of TRAM-34 in treating corneal fibrosis by measuring levels of pro-fibrotic genes, proteins, and cellular migration using real-time quantitative qPCR, Western blotting, and scratch assay, respectively. Cytotoxicity of TRAM-34 was tested with trypan blue assay, and pro-fibrotic marker expression was tested in KCa3.1-/-. Expression of KCa3.1 mRNA and protein was detected in all three layers of the human cornea. The KCa3.1-/- mice demonstrated significantly reduced corneal fibrosis and expression of pro-fibrotic marker genes such as collagen I and α-smooth muscle actin (α-SMA), suggesting that KCa3.1 plays an important role corneal wound healing in vivo. Pharmacological treatment with TRAM-34 significantly attenuated corneal fibrosis in vitro, as demonstrated in HCFs by the inhibition TGFβ-mediated transcription of pro-fibrotic collagen I mRNA and α-SMA mRNA and protein expression (p

PurposeWe tested the potential of bone morphogenic protein 7 (BMP7) and hepatocyte growth factor (HGF) combination gene therapy to treat preformed corneal fibrosis using established rabbit in vivo and human in vitro models.MethodsEighteen New Zealand White rabbits were used. Corneal fibrosis was produced by alkali injury. Twenty-four hours after scar formation, cornea received topically either balanced salt solution (BSS; n = 6), polyethylenimine-conjugated gold nanoparticle (PEI2-GNP)-naked plasmid (n = 6) or PEI2-GNP plasmids expressing BMP7 and HGF genes (n = 6). Donor human corneas were used to obtain primary human corneal fibroblasts and myofibroblasts for mechanistic studies. Gene therapy effects on corneal fibrosis and ocular safety were evaluated by slit-lamp microscope, stereo microscopes, quantitative real-time PCR, immunofluorescence, TUNEL, modified MacDonald-Shadduck scoring system, and Draize tests.ResultsPEI2-GNP-mediated BMP7+HGF gene therapy significantly decreased corneal fibrosis in live rabbits in vivo (Fantes scale was 0.6 in BMP7+HGF-treated eyes compared to 3.3 in -therapy group; P < 0.001). Corneas that received BMP7+HGF demonstrated significantly reduced mRNA levels of profibrotic genes: α-SMA (3.2-fold; P < 0.01), fibronectin (2.3-fold, P < 0.01), collagen I (2.1-fold, P < 0.01), collagen III (1.6-fold, P < 0.01), and collagen IV (1.9-fold, P < 0.01) compared to the -therapy corneas. Furthermore, BMP7+HGF-treated corneas showed significantly fewer myofibroblasts compared to the -therapy controls (83%; P < 0.001). The PEI2-GNP introduced >104 gene copies per microgram DNA of BMP7 and HGF genes. The recombinant HGF rendered apoptosis in corneal myofibroblasts but not in fibroblasts. Localized topical BMP7+HGF therapy showed no ocular toxicity.ConclusionsLocalized topical BMP7+HGF gene therapy treats corneal fibrosis and restores transparency in vivo mitigating excessive healing and rendering selective apoptosis in myofibroblasts.

Many factors contribute to patient survival in metastatic breast cancer and other late-stage cancers. It is doubtful that adjuvant therapy causes a worse survival outcome. This being said, should resistance develop following adjuvant therapy, the patient needs to be offered a first-line solution that is free of cross-resistance.

Background: Adjuvant treatment concepts have improved the 10-year cure rate of breast and colon cancer, but new treatments for metastatic disease have yielded only incremental benefit. If treatments for disseminated cancer were actually prolonging life rather than only increasing remission rates, this effect should have been documented over the last 30+ years. However, published data concerning advances in treatment for disseminated cancer have been contradictory. Patients and Methods: To add data-based information, we analyzed 2 sources: a regional population-based cancer registry (Hamburgisches Krebsregister, HKR), and a research cancer registry (Projektgruppe Internistische Onkologie, PIO). We compared the survival of several thousand patients with metastatic disease who received treatment only after dissemination with that of patients who received initial adjuvant therapy. Results: After adjuvant treatment, survival in patients with disseminated breast cancer is up to a third shorter than that of patients without adjuvant therapy. Conclusions: In accordance with published evidence, we conclude that ineffective adjuvant treatment shortens survival after documentation of metastatic disease. This is probably due to the elimination of chemo-sensitive tumor cells or to the induction of resistance in remaining micrometatases. This negative effect on survival after dissemination has been shown clearly for breast cancer and is also probable for cancer of the colon and other sites.

Based on the hypothesis that impaired glucose metabolism might be associated with survival outcomes independently of overt diabetes, we sought to investigate the prognostic value of routinely used glycemic parameters in a prospective study of breast cancer (BC) patients.Fasting blood glucose, insulin and HbA1c levels, and insulin resistance (assessed by the Homeostasis Model Assessment [HOMA] index) at diagnosis were evaluated in 286 nondiabetic BC patients (249 with primary cancer, 37 with metastatic) with respect to those parameters' possible associations with clinicopathological features and survival outcomes. As a control group, 143 healthy women matched in a 2:1 ratio for age, blood lipid levels, and body mass index were also investigated.Fasting blood glucose level (mean ± SD: 99 ± 26 vs. 85 ± 15 mg/dL), insulin level (median: 10.0 vs. 6.8 μIU/mL), and HOMA index (median: 2.2 vs. 1.4), but not HbA1c level, were significantly elevated in BC patients compared with control subjects. Receiver operating characteristics analysis showed comparable areas for blood glucose and insulin levels, and HOMA index (ranging from 0.668 to 0.671). Using a cutoff level of 13 μIU/mL, insulin had the best specificity (92%) and sensitivity (41%), was significantly associated with disease stage, and acted as a negative prognostic marker of progression-free survival (hazard ratio: 2.17; 95% confidence interval: 1.13-4.20) independently of menopausal status, disease stage, hormone receptor status, and human epidermal growth factor receptor 2 and Ki67 expression.These results suggest that insulin determination might provide prognostic information in BC and support the hypothesis that lifestyle and/or pharmacological interventions targeting glucose metabolism could be considered to improve survival outcome of selected BC patients.Pretreatment insulin levels may represent a biomarker of adverse prognosis in nondiabetic women with breast cancer, independently of other well-established prognostic factors (i.e., stage, hormone receptors, HER2/neu, and Ki67). This finding has important implications, because it provides the rationale for lifestyle or insulin-targeting pharmacologic interventions as a means of improving breast cancer outcomes not only in early stages, but also in advanced-stage breast cancer patients with aggressive tumor phenotypes (HER2-negative hormone-resistant, or triple-negative breast cancer), in which treatments are still challenging. The possibility of using insulin as a biomarker to guide insulin-targeted interventions also should be taken into account.

Shortening of survival after dissemination following adjuvant treatment is not understood well enough and is difficult to search for in public databases because of the lack of specific search terms. We suggest using the acronym ATRESS, for “adjuvant therapy-related shortening of survival,” in future literature concerning this issue.