Chandrakala Menon

Photofrin uptake in the tumor and normal tissues of patients receiving intraperitoneal photodynamic therapy

Purpose: A phase II trial of Photofrin-mediated i.p. photodynamic therapy shown in a previous report limited efficacy and significant acute, but not chronic, toxicity. A secondary aim of this trial and the subject of this report is to determine Photofrin uptake in tumor and normal tissues. Experimental Design: Patients received Photofrin, 2.5 mg/kg, i.v., 48 hours before debulking surgery. Photofrin uptake was measured by spectroflurometric analysis of drug extracted from tumor and normal tissues removed at surgery. Differences in drug uptake among these tissues were statistically considered using mixed-effects models. Results: Photofrin concentration was measured in 301 samples collected from 58 of 100 patients enrolled on the trial. In normal tissues, drug uptake significantly (P < 0.0001) differed as a function of seven different tissue types. In the toxicity-limiting tissue of intestine, the model-based mean (SE) Photofrin level was 2.70 ng/mg (0.32 ng/mg) and 3.42 ng/mg (0.24 ng/mg) in full-thickness large and small intestine, respectively. In tumors, drug uptake significantly (P = 0.0015) differed as a function of patient cohort: model-based mean Photofrin level was 3.32 to 5.31 ng/mg among patients with ovarian, gastric, or small bowel cancer; 2.09 to 2.45 ng/mg among patients with sarcoma and appendiceal or colon cancer; and 0.93 ng/mg in patients with pseudomyxoma. Ovarian, gastric, and small bowel cancers showed significantly higher Photofrin uptake than full-thickness large and/or small intestine. However, the ratio of mean drug level in tumor versus intestine was modest (≤2.31). Conclusions: Some selectivity is found in Photofrin uptake between tumor and normal tissues of the peritoneal cavity, but absolute differences in drug uptake relative to toxicity-limiting normal tissues (intestine) are small. This narrow differential in drug selectivity likely contributes to a narrow window in therapeutic application, which has been previously reported.

A Phase II Trial of Intraperitoneal Photodynamic Therapy for Patients with Peritoneal Carcinomatosis and Sarcomatosis

Purpose: A previous phase I trial of i.p. photodynamic therapy established the maximally tolerated dose of Photofrin (Axcan Pharma, Birmingham, AL)-mediated photodynamic therapy and showed encouraging efficacy. The primary objectives of this phase II study were to determine the efficacy and toxicities of i.p. photodynamic therapy in patients with peritoneal carcinomatosis and sarcomatosis. Experimental Design: Patients received Photofrin 2.5 mg/kg i.v. 48 hours before debulking surgery. Intraoperative laser light was delivered to the peritoneal surfaces of the abdomen and pelvis. The outcomes of interest were (a) complete response, (b) failure-free survival time, and (c) overall survival time. Photosensitizer levels in tumor and normal tissues were measured. Results: One hundred patients were enrolled into one of three strata (33 ovarian, 37 gastrointestinal, and 30 sarcoma). Twenty-nine patients did not receive light treatment. All 100 patients had progressed by the time of statistical analysis. The median failure-free survival and overall survival by strata were ovarian, 2.1 and 20.1 months; gastrointestinal cancers, 1.8 and 11.1 months; sarcoma, 3.7 and 21.9 months. Substantial fluid shifts were observed postoperatively, and the major toxicities were related to volume overload. Two patients died in the immediate postoperative period from bleeding, sepsis, adult respiratory distress syndrome, and cardiac ischemia. Conclusions: Intraperitoneal Photofrin-mediated photodynamic therapy is feasible but does not lead to significant objective complete responses or long-term tumor control. Heterogeneity in photosensitizer uptake and tumor oxygenation, lack of tumor specificity for photosensitizer uptake, and the heterogeneity in tissue optical properties may account for the lack of efficacy observed.

Expression of myosin isoforms in smooth muscle cells in the corpus cavernosum penis

Corpus cavernosum smooth muscle (CCSM) in the penis is unique in that it exhibits a high resting tone and, on stimulation, the muscle cells relax, allowing cavernous spaces to fill with blood, which results in an erection (tumescence). During detumescence, the muscle cells contract and return to the state of high resting tone. This study was undertaken to determine whether CCSM with these unique properties contains myosin isoforms typical of aorta or bladder smooth muscles, muscles that exhibit tonic and phasic characteristics, respectively. RT-PCR revealed that normal CCSM contains an SM2/SM1 mRNA ratio of 1.2:1 (similar to the rabbit aorta). Approximately 31% of the myosin heavy chain transcripts possess a 21-nt insert (predominant in bladder smooth muscle but not expressed in aorta) that encodes the seven-amino acid insert near the NH2-terminal ATP binding region in the head portion of the myosin molecule found in SMB, with the remaining mRNA being noninserted (SMA). Quantitative competitive RT-PCR revealed that the CCSM possesses approximately 4.5-fold less SMB than the bladder smooth muscle. Western blot analysis using an antibody specific for the seven-amino acid insert reveals that both SM1 and SM2 in the CCSM contain the seven-amino acid insert. Furthermore, SMB containing the seven-amino acid insert was localized in the CCSM by immunofluorescence microscopy using this highly specific antibody. The analysis of the expression of LC17 isoforms a and b in the CCSM revealed that it is similar to that of bladder smooth muscle. Thus the CCSM possesses an overall myosin isoform composition intermediate between aorta and bladder smooth muscles, which generally express tonic- and phasiclike characteristics, respectively. Two-dimensional gel electrophoresis showed a relatively low level (approximately 10%) of Ca2+-dependent light-chain (LC20) phosphorylation at the basal tone, which reaches approximately 23% in response to maximal stimulation. The presence of noninserted and inserted myosin isoforms with low and high levels of actin-activated ATPase activities, respectively, in the CCSM may contribute to the ability of the CCSM to remain in a state of high resting tone and to relax rapidly for normal penile function.Corpus cavernosum smooth muscle (CCSM) in the penis is unique in that it exhibits a high resting tone and, on stimulation, the muscle cells relax, allowing cavernous spaces to fill with blood, which results in an erection (tumescence). During detumescence, the muscle cells contract and return to the state of high resting tone. This study was undertaken to determine whether CCSM with these unique properties contains myosin isoforms typical of aorta or bladder smooth muscles, muscles that exhibit tonic and phasic characteristics, respectively. RT-PCR revealed that normal CCSM contains an SM2/SM1 mRNA ratio of 1.2:1 (similar to the rabbit aorta). Approximately 31% of the myosin heavy chain transcripts possess a 21-nt insert (predominant in bladder smooth muscle but not expressed in aorta) that encodes the seven-amino acid insert near the NH2-terminal ATP binding region in the head portion of the myosin molecule found in SMB, with the remaining mRNA being noninserted (SMA). Quantitative competitive RT-PCR revealed that the CCSM possesses ∼4.5-fold less SMB than the bladder smooth muscle. Western blot analysis using an antibody specific for the seven-amino acid insert reveals that both SM1 and SM2 in the CCSM contain the seven-amino acid insert. Furthermore, SMB containing the seven-amino acid insert was localized in the CCSM by immunofluorescence microscopy using this highly specific antibody. The analysis of the expression of LC17isoforms a and b in the CCSM revealed that it is similar to that of bladder smooth muscle. Thus the CCSM possesses an overall myosin isoform composition intermediate between aorta and bladder smooth muscles, which generally express tonic- and phasiclike characteristics, respectively. Two-dimensional gel electrophoresis showed a relatively low level (∼10%) of Ca2+-dependent light-chain (LC20) phosphorylation at the basal tone, which reaches ∼23% in response to maximal stimulation. The presence of noninserted and inserted myosin isoforms with low and high levels of actin-activated ATPase activities, respectively, in the CCSM may contribute to the ability of the CCSM to remain in a state of high resting tone and to relax rapidly for normal penile function.

Tumor necrosis factor-alpha damages tumor blood vessel integrity by targeting VE-cadherin.

Background:Isolated limb perfusion using high-dose human tumor necrosis factor-alpha with melphalan is effective therapy for bulky extremity in-transit melanoma and sarcoma. Objective:While it is widely accepted that melphalan is a DNA alkylating agent, the mechanism of selective antitumor effect of tumor necrosis factor-alpha is unclear. Methods and Results:Electron microscopic analyses of human melanoma biopsies, pre- and post-melphalan perfusion, showed that the addition of tumor necrosis factor-alpha caused gapping between endothelial cells by 3 to 6 hours post-treatment followed by vascular erythrostasis in treated tumors. In human melanoma xenografts raised in mice, tumor necrosis factor-alpha selectively increased tumor vascular permeability by 3 hours and decreased tumor blood flow by 6 hours post-treatment relative to treated normal tissue. In an in vitro tumor endothelial cell model, tumor necrosis factor-alpha caused vascular endothelial adherens junction protein, VE-cadherin, to relocalize within the cell membrane away from cell-cell junctions leading to gapping between endothelial cells by 3 to 6 hours post-treatment. Phosphotyrosinylation was a prerequisite for movement of VE-cadherin away from endothelial cell junctions and for gapping between endothelial cells. Clinical isolated limb perfusion tumor specimens, at 3 hours postperfusion, showed a discontinuous and irregular pattern of VE-cadherin expression at endothelial cell junctions when compared with normal (skin) or pretreatment tumor tissue. Conclusions:Together, the data suggest that tumor necrosis factor-alpha selectively damages the integrity of tumor vasculature by disrupting VE-cadherin complexes at vascular endothelial cell junctions leading to gapping between endothelial cells, causing increased vascular leak and erythrostasis in tumors. VE-cadherin appears to be a potentially good target for selective antitumor therapy.

Contractile protein changes in urinary bladder smooth muscle following outlet obstruction.

Force production by the bladder body smooth muscle and relaxation of the bladder outlet, required for bladder emptying, are regulated by Ca2+ via myosin light chain (MLC) phosphorylation by a Ca2+-calmodulin-dependent MLC kinase (for review, 1–3). Besides this mode of regulation, evidence from most laboratories, including ours, supports the existence of a thin-filament-mediated regulation due to protein-protein interaction by actin-binding proteins acting in concert with tropomyosin and a calcium-binding protein, most likely, calmodulin. Caldesmon (CaD), a thin-filament-associated protein, inhibits the ac-tin-myosin interaction and actomyosin ATPase, and this inhibition is reversed by calmodulin in the presence of Ca2+.3–5 Urinary bladder outlet obstruction interferes with the ability of the bladder to empty its contents, thereby inducing changes in the bladder wall smooth muscle cells which enable the bladder to produce the increased contractile force required to expel urine through the obstructed urethra. In the initial phases of outlet obstruction, there is transient decompensation of the bladder smooth muscle, which initiates the molecular events that lead to hypertrophy of the detrusor smooth muscle cells enabling it to generate and maintain force. However, continuation of the outlet obstruction induces molecular, cellular, and structural alterations in the muscle cells of the bladder wall, leading ultimately to decreased compliance and impaired emptying. The ability of smooth muscles to compensate for increased functional demand is associated with alterations in the expression of proteins in the thick- and thin-filaments.

Validity of Subject Self-Report for Acne

Background/Objective: Acne prevalence studies often use subject self-report as data source. Our aim was to evaluate the validity of acne self-report. Methods: Responses of university students to an acne questionnaire were compared to the trained observer’s concurrent examination of acne. The validity of self-report was measured by sensitivity, specificity, positive predictive value and negative predictive value. Agreement was measured by Cohen’s kappa and correct classification percentage. Results: The sensitivity of self-report was 0.55 (95% CI 0.47–0.63), the specificity was 0.72 (95% CI 0.63–0.80), the positive predictive value was 0.70 (95% CI 0.61–0.78), and the negative predictive value was 0.57 (95% CI 0.49–0.65). Cohen’s kappa was 0.26 (95% CI 0.15–0.38) and correct classification percentage was 63. Conclusions: Validity of self-report was moderate at best and agreement was fair, indicating that college students could not accurately report that they have acne. This is likely not sufficient for clinical or research activities or to assure that individuals who self-guide their acne therapy actually have acne.

Tumoricidal activity of high-dose tumor necrosis factor-α is mediated by macrophage-derived nitric oxide burst and permanent blood flow shutdown

This study investigates the role of tumor nitric oxide (NO) and vascular regulation in tumor ulceration following high‐dose tumor necrosis factor‐α (TNF) treatment. Using TNF‐responsive (MethA) and nonresponsive (LL2) mouse tumors, tumor NO concentration was measured with an electrochemical sensor and tumor blood flow by Doppler ultrasound. Mice were also pretreated with a selective inducible nitric oxide synthase (iNOS) inhibitor, 1400 W. Tumors harvested from TNF‐treated mice were cryosectioned and immunostained for murine macrophages, or/and iNOS. MethA tumor‐bearing mice were depleted of macrophages. Pre‐ and post‐TNF tumor NO levels were measured continuously, and mice were followed for gross tumor response. In MethA tumors, TNF caused a 96% response rate, and tumor NO concentration doubled. Tumor blood flow decreased to 3% of baseline by 4 hr and was sustained at 24 hr and 10 days post‐TNF. Selective NO inhibition with 1400 W blocked NO rise and decreased response rate to 38%. MethA tumors showed tumor infiltration by macrophages post‐TNF and the pattern of macrophage immunostaining overlapped with iNOS immunostaining. Depletion of macrophages inhibited tumor NO increase and response to TNF. LL2 tumors had a 0% response rate to TNF and exhibited no change in NO concentration. Blood flow decreased to 2% of baseline by 4 hr, recovered to 56% by 24 hr and increased to 232% by 10 days. LL2 tumors showed no infiltration by macrophages post‐TNF. We conclude that TNF causes tumor infiltrating, macrophage‐derived iNOS‐mediated tumor NO rise and sustained tumor blood flow shutdown, resulting in tumor ulceration in the responsive tumor.

Mature CD83+ dendritic cells infected with recombinant gp 100 vaccinia virus stimulate potent antimelanoma T cells

BackgroundMature dendritics cells (DCs) are potent antigen-presenting cells that activate naive T lymphocytes and initiate cellular immune responses. The ability of CD83+ mature DCs infected with vaccinia virus encoding the gp 100 melanoma transgene (rV-gp 100) to stimulate an antimelanoma CD8+ T-cell response was investigated.MethodsMonocyte-derived immature or CD83+ mature DCs were infected with rV-gp100. The activation state of the DCs and the expression of gp 100 protein were evaluated by flow cytometry. The reactivity of antimelanoma CD8+ T cells was confirmed by measuring specific interferon γ secretion by using enzyme-linked immunosorbent assay in a mixed-tumor lymphocyte culture.ResultsBoth immature and CD83+ mature DCs expressed gp 100 protein when the DCs were infected with rV-gp 100. Calcium-signaling agents were required to induce maturation of both infected and nonifected immature DCs. Only rV-gp100-infected CD83+ DCs induced CD8+ T cells, after a single stimulation that recognized both peptide-pulsed target cells to multiple gp 100 epitopes and a melanoma cell line that endogenously expressed gp 100 antigen.ConclusionsCD83+ DCs transduced with rV-gp 100 are capable of generating a strong CD8+ T-cell response against melanoma tumor cells. Expression of melanoma antigens by mature DCs offers the potential advantage of presenting multiple endogenously processed T-cells epitopes and using multiple HLA restriction elements for antimelanoma vaccine therapy.

Analysis of Tumor Thiol Concentrations: Comparison of Flow Cytometric With Chemical and Biochemical Techniques

The importance of glutathione (GSH) in contributing to cancer therapy resistance is well established. Various advantages may accrue from the ability to determine the distribution of GSH content in individual tumor cells disaggregated from solid tumors using flow cytometric techniques compared with biochemical or chemical measurements of the average GSH level in bulk tissue samples. The flow cytometric technique requires a thiol-reactive fluorescent adduct which is stable and which can differentiate cellular GSH from protein thiols. Thiol-reactive compounds specific for GSH require facilitated conjugation by endogenous cellular enzymes, but such compounds have not been found to accurately monitor GSH in human cells. Compounds which react generally with all thiols require a GSH-depleted calibration control to assess GSH vs. non-GSH components of fluorescent-adduct formation. Our report addresses this question, and compares three different thiol assays in several cell lines. We have found a simple way to control for non-GSH adduct formation. This involves a selective permeabilization process to release low molecular weight adducts (dominated by GSH and cysteine). In application to the desired goal of assessing the distribution of GSH in cells disaggregated from tumors, we have identified a problem with cell-line-specific thiol loss during the tumor cell disaggregation process.