John Mcmichael

Nerve growth factor (NGF) has novel antidepressant-like properties in rats

Nerve growth factor, a neurotrophin, may have other functions, including a role in depressive disorders. The present study sought to determine whether NGF would (1) have antidepressant-like effects and (2) behave similarly to or differently from other well-recognized antidepressants. Over a broad dose-range, NGF reduced the exaggerated swim test immobility exhibited by the Flinders Sensitive Line (FSL) rats, but at a standard dose of 40 ng/ml, it was not as effective as desipramine (DMI, 5 mg/kg). The low social interaction behavior and locomotor activity of the FSL rats were less affected by NGF than was the immobility. Acute treatment with NGF did not induce c-fos expression in brain regions known to be activated by other acute antidepressants. The fact that chronic treatment with DMI blunted the corticosterone response to fluoxetine was replicated in this study. However, chronic treatment with NGF did not alter this response. Similarly, chronic treatment with fluoxetine blunted 5-HT(1A) and 5-HT(2A) receptor-mediated responses, whereas chronic treatment with NGF was without effect. Thus, NGF has antidepressant-like effects but does not appear to have biochemical actions typical of other antidepressants.

The Use of Streptolysin O for the Treatment of Scars, Adhesions and Fibrosis: Initial Investigations Using Murine Models of Scleroderma

Diseases and conditions involving the deposition of excessive amounts of collagen include scleroderma, fibrosis, and scar and surgical adhesion formation. Diseases such as scleroderma may result from acute and chronic inflammation, disturbances in the normal parenchymal area, and activation of fibroblasts. ML-05, a modified form of the hemolytic and cytotoxic bacterial toxin, streptolysin O, is being developed for the treatment of such collagen-related disorders. At sublytic concentrations in vitro, ML-05 was shown to activate CD44 expression. This may modulate production of collagen, hyaluronate, and their associated enzymes to allow a restoration of normal extracellular matrices within tissues. More importantly, ML-05 appeared to decrease skin collagen levels in two in vivo models of collagen disorders, the tight skin mouse (Tsk) model of scleroderma, and the bleomycin-induced mouse skin fibrosis model. In the Tsk model, levels of hydroxyproline (a measure of total collagen) decreased by 25% in the Tsk+ML-05 treatment group relative to the Tsk+saline control group over a 3-month period. In the bleomycin-induced skin fibrosis study, hydroxyproline levels decreased from 15–22% over a 6-week period in a bleomycin-induced ML-05 treatment group (relative to levels in a bleomycin-induced, untreated control group). Hydroxyproline levels in samples from this treatment group were only slightly greater than levels in an uninduced control group at 8 weeks. Thus, ML-05 treatment appeared to reduce collagen levels in two separate mouse skin fibrosis models, one genetically based and the other chemically induced.

Enhancement of radiosensitivity of the MCF-7 breast cancer cell line with human chorionic gonadotropin.

Secretion of human chorionic gonadotropin (hCG) during pregnancy induces differentiation of the mammary gland, thereby making breast tissue less susceptible to carcinogenesis. HCG binds to specific hCG receptors on mammary epithelial cells inducing changes in gene expression that can inhibit cell proliferation and, therefore, interfere with tumorigenesis. Since breast cancer cells also contain a relatively high level of the hCG receptor, hCG has potential as a therapeutic agent. We postulated that hCG might also enhance the radiosensitivity of breast cancer cells and, therefore, be useful as an adjunctive therapy. In the present study, MCF-7 breast cancer cells grown in cell culture were treated with hCG (0.2–5 IU/ml) for 24 h prior to exposing the cells to 0 Gy, 3 Gy, 4 Gy, or 5 Gy of radiation. Following irradiation, the MCF-7 cells were incubated either in the presence or absence of hCG. Cell survival was monitored with an MTT assay 1 day, 4 days, and 7 days after irradiation. All of the concentrations of hCG tested enhanced radiosensitivity of MCF-7 cells. The maximum enhancement occurred with MCF-7 cells that had been exposed to 2 IU/ml of hCG for at least 24 h prior to irradiation with 4 Gy. The use of higher concentrations of hCG or a higher dose of radiation did not increase the enhancement effect. Treatment of MCF-7 cells with hCG for only 24 h was sufficient to achieve the maximum effect. However, maintaining the cells in hCG beyond 24 h increased the effectiveness of the lowest hCG concentration. Using a linear-quadratic equation to analyze the data, we determined that the use of hCG would result in an 8–10% reduction in MCF-7 cell survival at a dose of 2 Gy, a typical dose used in conventional cancer therapy.

Inhibition of human breast cancer Matrigel invasion by Streptolysin O activation of the EGF receptor ErbB1.

Streptolysin O (SLO) is a protein cytotoxin derived from Group A beta-hemolytic streptococci that associates with membranes and permeabilizes cells. Oxidation inactivates SLO, eliminating the characteristic hemolytic and cytotoxic activities. However, oxidized SLO produces beneficial therapeutic effects in vivo on scleroderma, scar formation and wound healing. Here we report that oxidized SLO also significantly inhibited invasion by human metastatic breast cancer MDA-MB-231 cells through Matrigel in an in vitro model of metastatic disease. This dose-dependent response corresponded to selective SLO activation of epidermal growth factor receptor (EGFR) ErbB1. SLO and EGF were equally selective in activation of EGFR, but EGF elicited larger relative increases in phosphorylation at various sites, especially pronounced for Tyr845. Addition of SLO did not affect either ERK1/2 or Akt kinases and altered the expression of only 10 of 84 metastasis-related genes in MDA-MB-231 cells. Neither SLO nor EGF promoted growth of several human breast cancer cell lines. Knockdown of EGFR by siRNA ablated the inhibitory effect of SLO on cancer cell invasion, showing SLO selectively activated ErbB1 kinase to reduce invasion without increasing cell growth. The results suggest SLO might have promise as a new therapy to inhibit metastasis.

Effects of Streptolysin O on Extracellular Matrix Gene Expression in Normal Human Epidermal Keratinocytes

ML-05 is a non-hemolytic form of streptolysin O, the membrane-damaging extracellular toxin produced by certain streptococci. ML-05 stimulates keratinocyte migration and proliferation in wound-healing scratch assays and promotes wound healing in a human skin organ culture wound model. Pathway-focused DNA microarrays were used to elucidate ML-05s mechanism of action in wound healing processes. Normal human epidermal keratinocytes (NHEK) were treated with varying concentrations of ML-05 for 24 hours, followed by RNA extraction and cRNA production. Gene expression profiling utilized microarrays containing nucleic acid probes for 113 extracellular matrix (ECM) genes. Microarrays yielded 6 upregulated and 4 downregulated genes with ≥2-fold changes and p<0.05 in t-tests. Quantitative real-time polymerase chain reactions (qPCR) were used to verify gene regulation. Upregulated genes of interest were VCAN (formerly CSPG2, encoding versican), CD44 (encoding hyaluronan receptor), ICAM1 (encoding intercellular adhesion molecule-1) and CTGF (encoding connective tissue growth factor). All four upregulated genes encode proteins involved in promoting keratinocyte migration and proliferation. Downregulated genes of interest were MMP9 (encoding matrix metalloproteinase 9) and SPP1 (encoding osteopontin). ML-05 may enhance wound healing through the expression of specific genes encoding proteins capable of promoting keratinocyte migration, proliferation, and other activities related to maintaining ECM structure and function.

Transcriptional Changes in Cancer Cells Induced by Exposure to a Healing Method

Energy healing, or healing with intent, is a complementary and alternative medicine therapy reported to be beneficial with a wide variety of conditions. We are developing a delivery technology for a method previously tested in mouse models with solid tumors (the Bengston method) independent of the presence of a healer. The goal of this study was to assess whether stored or recorded energy has an impact on breast cancer cells in vitro, using energy-charged cotton and electromagnetic recording of healers practicing the method. Expression of genes involved in cancer and inflammation pathways was measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Treatment of cells using energy-charged cotton resulted in statistically significant changes <1.5-fold. In cells exposed to an electromagnetic recording, 37 genes of 167 tested showed a >1.5-fold change when compared to the control, and 68 genes showing statistically significant fold changes. Two genes, ATP citrate lyase (ACLY) and interleukin 1β (IL-1β), were consistently downregulated at 4 and 24 hours of exposure to the recording, respectively, in 3 independent experiments. Both ACLY and IL-1β were also downregulated in cells exposed to a hands-on delivery of the method, suggesting these 2 genes as potential markers of the healing method.

Combination histamine and serotonin treatment after simulated childbirth injury improves stress urinary incontinence.

Histamine and serotonin‐related pharmaceuticals have the potential to modulate micturition and continence. The aim of this study was to determine if treatment with histamine and/or serotonin improves stress urinary incontinence (SUI) in female rats.

Serotonin and Histamine Therapy Increases Tetanic Forces of Myoblasts, Reduces Muscle Injury, and Improves Grip Strength Performance of Dmdmdx Mice

Duchenne muscular dystrophy (DMD) is a recessive X-linked fatal disorder caused by a mutation in the dystrophin gene. Although several therapeutic approaches have been studied, none has led to substantial long-term effects in patients. The aim of this study was to test a serotonin and histamine (S&H) combination on human skeletal myoblasts and Dmdmdx mice for its effects on muscle strength and injury. Normal human bioartificial muscles (BAMs) were treated, and muscle tetanic forces and muscle injury tests were performed using the MyoForce Analysis System. Dmdmdx mice, the murine model of DMD, were administered serotonin, histamine, or S&H combination twice daily for 6 weeks, and functional performance tests were conducted once a week. The S&H combination treatment caused significant increases in tetanic forces at all time points and concentrations tested as compared to the saline controls. Dose response of the BAMs to the treatment demonstrated a significant increase in force generation at all concentrations compared to the controls after 3 to 4 days of drug treatment. The highest 3 concentrations had a significant effect on lowering contractile-induced injury as measured by a reduction in the release of adenylate kinase. Histamine-only and S&H treatments improved grip strength of Dmdmdx mice, whereas serotonin-only treatment resulted in no significant improvement in muscle strength. The results of this study indicate that S&H therapy might be a promising new strategy for muscular dystrophies and that the mechanism should be further investigated.