Accumulation of 5-aminolevulinic acid-induced protoporphyrin IX in normal and neoplastic human endometrial epithelial cells

The aim of this study was to evaluate 5-Aminolevulinic acid (ALA)-induced fluorescence of normal and neoplastic endometrial epithelial cells for diagnosis and photodynamic treatment. Fluorescence of ALA-induced PpIX in vitro was measured by flow cytometry in two different human endometrial adenocarcinoma cell lines and in normal cells cultivated from fresh endometrial tissue of three premenopausal patients. The cells were analysed after incubation with different concentrations of ALA during 3, 6, or 24 hours. Both tumor cell lines showed a statistically significant higher fluorescence of PpIX than normal epithelial cells after incubation with 1 mg ALA per ml medium during 24 hours. The well-differentiated cancer cells produced significantly more PpIX than the poorly differentiated cancer cells. Relative PpIX intensity of the two cancer cell lines correlated with cell proliferation rate as measured by the doubling times of the cells. Higher accumulation of Pp IX in neoplastic endometrium compared to normal endometrial epithelial cells may provide targeted biopsies and selective photodynamic destruction of neoplastic micro-lesions.     

Where does venous reflux start?

To examine the potential of using photodynamic therapy (PDT) in condylomata, we studied the distribution and kinetics of protoporphyrin IX (PpIX) formation in condylomata acuminata and adjacent normal skin after topical application of 5-aminolaevulinic acid (ALA). PpIX fluorescence spectra were measured hourly in vivo after ALA application. After gross fluorescence imaging, the lesions were biopsied, and fluorescence microscopy was performed. All three PpIX fluorescence detection modalities suggested selectivity of PpIX formation in condylomata after topical ALA application. In 17 of 25 condylomata, there was significantly greater fluorescence compared with adjacent normal skin. The greatest lesional to normal skin fluorescence ratios occurred after 2 h. The most likely mechanism for increased lesional PpIX formation in condylomata is enhanced stratum corneum permeability. Based on our results, ALA/PDT is a potential field therapy for condylomata. PpIX fluorescence imaging after ALA application may also be useful for localizing condylomata prior to treatment.     

Production of protoporphyrin IX induced by 5-aminolevulinic acid in transplanted human colon adenocarcinoma of nude mice can be increased by ultrasound

BALB/c nude mice bearing WiDr human colon adenocarcinoma were used to determine the effect of ultrasound on the production of 5-aminolevulinic acid (ALA)-induced protoporphyrin IX (PpIX) both in the tumors and in skin overlying the tumors. Ultrasound (1 MHz) with pulsed irradiation at an average intensity of 3 W/cm2 was given 10 min to the tumor area 10 min after administration of ALA (20% in an oil-in-water emulsion applied topically on the surface of the tumor for 30 min to 3 hr). An approximately 45% increase in the amount of PpIX produced by ALA in the tumors was obtained within 1 to 2 hr following ultrasound treatment. In particular, 1 hr after ultrasound treatment, the amount of PpIX in the tumors was at the same level as that 3 hr after ALA application alone. However, pulsed ultrasound irradiation for 5 min or continuous irradiation for 5 or 10 min had no significant effect on the production of PpIX by the tumor 1 hr after topical ALA application. Furthermore, in most cases, the amount of PpIX in the tumors was significantly decreased when ultrasound was given immediately before ALA application. There was no significant change in the ratio of the amount of PpIX in tumor to that in skin after ultrasound treatment. Most likely, the distribution of PpIX fluorescence in the tumors treated with ultrasound was more homogeneous than that in the tumors given ALA only. Our results provide a theoretical basis for possible clinical use of ultrasound-combined ALA or ALA based photodynamic therapy.     

Investigation of specimen mislabelling in paraffin-embedded tissue using a rapid, allele-specific, PCR-based HLA class II typing method

Topical application of 5-aminolevulinic acid (5-ALA), with subsequent synthesis of protoporphyrin IX (PPIX), is a novel outstanding procedure for photodynamic treatment. So far, clinical experience has been reported with creams containing 5-ALA for the therapy of skin cancer, oral application for the treatment of gastrointestinal disease and intravesical instillation of 5-ALA solutions for fluorescence detection of superficial bladder cancer. Inhalation of 5-ALA for the staining of bronchial malignancies is a preferred method in clinical pulmonology. Since no adverse reaction was observed in lung function in a canine following inhalation of 5-ALA in increasing concentrations, clinical applications were performed. Seven patients with positive or suspicious sputum cytology, but negative white light bronchoscopy, received 5-10 wt.% 5-ALA in NaCl solution by means of a medical nebulizer. No side effects were observed during and after 5-ALA inhalation. After a period of 3 h, patients underwent fluorescence bronchoscopy using violet light for fluorescence excitation and an optical multichannel analyzer for fluorescence detection in situ. The results showed fluorescence spectra which could be related to PPIX induced by 5-ALA in the bronchial mucosa. The fluorescence intensity was sufficiently high for video imaging using a target integrating color CCD camera adapted to the flexible bronchoscope. Carcinoma in situ, as well as dysplasias, showed a clear positive fluorescence. A correlation of fluorescence contrast with histology on 30 biopsies revealed a high sensitivity, but a specificity below 50%. Improvements in light and drug dosimetry will form the basis for further clinical trials.     

Human microsporidiosis in the acquired immunodeficiency syndrome

BACKGROUND AND OBJECTIVE: Photodynamic therapy (PDT) using 5-aminolevulinic acid (ALA)-induced protoporphyrin IX (PpIX) for sensitization is a promising treatment for carcinoma in situ and diffuse premalignant changes of the bladder. We studied the biodistribution of PpIX in a range of tissues with oral and intravesical routes of administration of ALA and compared the photodynamic effects on bladder and skin. STUDY DESIGN/MATERIALS AND METHODS: Normal Wistar rats were given oral or intravesical ALA and PpIX levels in the liver, kidney, skin, and bladder measured by fluorescence microscopy on tissue sections. At the time of maximum PpIX levels, the bladder and skin on the back were illuminated with light at 630 nm and the PDT effects compared. RESULTS: PpIX fluorescence in the urothelium after 200 mg/kg given intravesically was comparable to that found after 100 mg/kg orally. The ratio of PpIX levels between the urothelium and the underlying muscle was the same for both routes of administration, although there appeared to be more selectivity of urothelial PDT necrosis after intravesical administration. Skin photosensitization was greater after oral ALA, the epidermal PpIX level being three times higher than after intravesical administration for comparable urothelial levels and the PDT effect being more marked. CONCLUSIONS: Intravesical instillation is preferable to oral administration of ALA for PDT ablation of the urothelium of the rat bladder without damage to the underlying tissue layers and for minimizing skin photosensitivity. The technique is now ready for clinical trials.     

Noninvasive diagnosis of oral neoplasia based on fluorescence spectroscopy and native tissue autofluorescence

OBJECTIVE: To evaluate the clinical potential of fluorescence spectroscopy (a noninvasive technique for assessing the chemical and morphologic composition of tissue) for in vivo detection of oral cavity neoplasia. DESIGN: A fluorescence spectroscopy system recorded spectra from oral cavity sites in 8 healthy volunteers and in 15 patients with premalignant or malignant oral cavity lesions at 337-, 365-, and 410-nm excitation wavelengths in the emission range of 350 to 700 nm. Fluorescence peak intensities and spectral line shapes were compared and diagnostic algorithms were developed to distinguish normal sites from abnormal sites. SETTING: The head and neck cancer clinic at a tertiary referral center in Houston, Tex. RESULTS: Differences were found in spectra from normal, dysplastic, and malignant oral mucosa. The fluorescence intensity of normal mucosa was greater than that of abnormal areas. In addition, the ratio of red region (635-nm) to blue region (455-490-nm) intensities was greater in abnormal areas. Diagnostic discrimination was achieved when test site spectra were compared with spectra from a normal site in the same patient. One diagnostic algorithm based on spectra at 337 nm gave a sensitivity of 88% and a specificity of 100%. CONCLUSIONS: Consistent differences exist between the fluorescence spectra of abnormal and normal oral mucosa. Therefore, fluorescence spectroscopy has the potential to improve the noninvasive diagnosis of oral cavity neoplasia. Further studies will better define the role of this technique in the detection of premalignant and early oral cancer lesions.     

Comparison of photodynamic therapy with cryotherapy in the treatment of Bowen's disease

The efficacy and suitability of photodynamic therapy (PDT) was compared with that of cryotherapy in the treatment of 40 lesions of Bowen's disease. Lesions were randomized to receive either cryotherapy with liquid nitrogen, or PDT using a portable desktop lamp incorporating a 300 W xenon short arc discharge source. A porphyrin precursor, 5-aminolaevulinic acid (5-ALA), was applied topically 4 h before irradiation in the PDT group. Each lesion received 125 J/cm2 at a fluence rate of 70 mW/cm2. All patients were reviewed at 2-monthly intervals and treatments repeated if required. Cryotherapy produced clearance in 10 of 20 lesions after one treatment, the remaining 10 lesions requiring two or three treatment applications. PDT resulted in clearance of 15 of 20 lesions after one treatment and of the remaining five lesions after a second treatment. The probability that a lesion cleared after one treatment was greater with PDT than cryotherapy (P < 0.01). Cryotherapy was associated with ulceration (five of 20), infection (two of 20) and recurrent disease (two of 20); no such complications occurred following PDT. PDT using a non-laser light source and topical 5-ALA appears to be at least as effective as cryotherapy in the treatment of Bowen's disease with fewer adverse effects.     

Quantitative studies of the kinetics of 5-aminolaevulinic acid-induced fluorescence in bladder transitional cell carcinoma

Photodynamic therapy is a potential treatment for superficial bladder cancer that utilizes photosensitizer drugs, which are activated by light to cause tissue destruction. However, first-generation photosensitizers cause prolonged phototoxicity, have poor tumour specificity and can accumulate within detrusor muscle, resulting in permanent loss of bladder capacity following treatment. A newer drug, called 5-aminolaevulinic acid (ALA), generates a sensitizer called protoporphyrin IX (PpIX) in situ and has been shown, qualitatively, to be more tumour specific. The fluorescence kinetics of ALA-induced PpIX was investigated in patient biopsies of bladder tumour, normal urothelium and detrusor muscle, both in vitro after incubation of specimens in ALA-rich culture medium for various times and in vivo after instillation of intravesical ALA before endoscopic resection. The fluorescence in tumour tissue was twice that of normal urothelium in vitro and up to tenfold in vivo. There was little ALA-induced fluorescence in detrusor muscle, both in vitro and in vivo. Most importantly, no patients experienced phototoxicity or other adverse events following intravesical instillation of ALA.     

In vivo fluorescence kinetics of porphyrins following intravesical instillation of 5-aminolaevulinic acid in normal and tumour-bearing rat bladders

The fluorescence kinetics of protoporphyrin IX (PPIX) following intravesical instillation of 5-aminolaevulinic acid (5-ALA) have been studied in vivo in a rat bladder tumour model. 5-ALA dissolved in NaHCO3 was intravesically instilled for 60 min in tumour-bearing and normal bladders of Wistar rats. The fluorescence was excited with the violet lines of a Kr(+)-laser and recorded in vivo by means of a fibre coupled optical multichannel analyser. The fluorescence emission bands of PPIX at lambda = 636 nm and lambda = 708 nm were detected in normal and tumorous urothelium after only 30 min. The maximum fluorescence intensity was obtained in tumorous and normal urothelium 3-4 h after instillation. The ratio of the fluorescence intensity in tumorous to normal urothelium decreased continuously from four to about two during the time range of 6 h. PPIX fluorescence following 5-ALA instillation could also be observed in kidney and liver. Fluorescence from further porphyrin species with emission bands at lambda = 617 nm and lambda = 682 nm was detected in the bladder, indicating an efflux of hydrophilic porphyrins from the hepatic pathway.     

Influence of diagnostic and treatment factors in the population pharmacokinetics of gentamicin

STUDY OBJECTIVES: To determine the feasibility of macroscopic visualization of small ovarian cancer metastases in vivo by fluorescence after intravenous administration of 5-aminolevulinic acid (ALA); to assess the time after drug injection when fluorescence of small metastases is maximum; and to correlate macroscopic in vivo fluorescence with both microscopic ex vivo fluorescence and histologic findings. DESIGN: Controlled animal study (Canadian Task Force classification I). SETTING: University-based facility. SUBJECTS: Twenty-four healthy, female Fischer rats. INTERVENTION: Diffuse peritoneal metastatic cancer was induced in Fischer 344 rats by intraperitoneal injection of 1 million syngeneic ovarian cancer cells (NuTu-19). Four weeks after induction ALA100 mg/kg was injected intravenously, and diagnostic laparotomy was performed 1, 3, 6, or 9 hours thereafter. MEASUREMENTS and MAIN RESULTS: The peritoneal cavity was illuminated with the Wood's lamp (ultraviolet light). Fluorescence was determined by direct visualization and compared with a calibrated fluorescent disk. Tissues were collected, sectioned, and examined by fluorescence and conventional light microscopy. Within 1 to 3 hours after intravenous injection of ALA, in vivo fluorescence of tumor nodules (diameter 0.4-5.0 mm) was macroscopically visible. Tumor-free peritoneum did not show fluorescence and was significantly distinguishable from cancer nodules. Fluorescence from intestinal tissues was comparable with tumor nodules. Microscopic fluorescence analysis showed similar values for tumor nodules and peritoneum. Stained histologic specimens of peritoneal surface revealed a superficial layer of cancer cells responsible for fluorescence. The time course of the fluorescence curve in the intestine peaked twice, at 1 and 6 hours after ALA injection. Macroscopically fluorescing nodules were histology confirmed as malignant. CONCLUSIONS: Fluorescence detection of small cancer nodules after intravenous injection of ALA is feasible for nodules smaller than 0.5 mm on the peritoneum. One to 3 hours after drug injection is optimal for diagnosis of metastases.     

Feasibility of photodynamic therapy using endogenous photosensitization for colon cancer

A novel approach to photodynamic therapy (PDT) involves endogenous photosensitization by the oral administration of delta-aminolevulinic acid (ALA), a naturally occurring substance that is the precursor of protoporphyrin IX (PpIX). A 60-year-old man with adenocarcinoma of the sigmoid colon received ALA, 60 mg/kg by mouth. Six hours later, when the plasma level of PpIX had peaked, the tumor was exposed locally to red light at 633 nm to activate PpIX. Endoscopy and biopsy findings subsequent to this treatment showed unequivocal visible changes and necrosis. Six months later, the patient again underwent successful treatment without adverse effects. This report suggests a role for PDT using endogenous photosensitization in certain circumstances involving adenocarcinoma of the large intestine.     

An open, randomized study of ketoprofen in patients in surgery for Achilles or patellar tendinopathy

OBJECTIVE: To assess the concentration of ketoprofen, after topical plaster application, in various tissues in relation to plasma levels in 60 patients undergoing surgery for Achilles or patellar tendinopathy; and to analyze whether tissues act as a reservoir of ketoprofen, by evaluating tissue concentrations in relation to plasma concentration at various time points after removal of the plaster. No attempt was made to study the clinical effect of treatment per se. METHODS: In random order to patients applied 30 mg plasters once daily for 5 consecutive days (n = 30), or took a single oral dose 50 mg (n = 30) before surgery. Tissue samples of skin, subcutaneous fat, tendon sheath, and tendon, and plasma were collected intraoperatively at 0, 6 and 14 hours after removal of the 5th plaster, and at 2, 6, and 14 hours after oral intake. Ketoprofen concentration was determined by a validated GC/MS method. The low limit of quantification was 0.5 ng/ml plasma and 0.5 ng/50 mg tissues. RESULTS: High concentrations of ketoprofen were observed in fat, tendon sheath, and tendon after topical applications, whereas plasma levels of ketoprofen were low. CONCLUSION: Ketoprofen attains high concentrations in subcutaneous tissues after multiple topical applications. Subcutaneous tissues appear to act as a reservoir of ketoprofen.     

In vitro studies on the potential use of 5-aminolaevulinic acid-mediated photodynamic therapy for gynaecological tumours

Results are reported on the sensitivity of various gynaecological tumour cell lines to 5-aminolaevulinic acid-induced protoporphyrin IX-sensitised photodynamic therapy (ALA-PDT) in vitro. All cell lines tested accumulated ALA-induced protoporphyrin IX (PpIX) and demonstrated good sensitivity to ALA-PDT. Localisation of PpIX in the mitochondria was demonstrated by fluorescence microscopy. Subcellular damage following ALA-PDT was observed using transmission electron microscopy. This damage was localised initially to the mitochondria, with damage to membranes and the nucleus and complete loss of intracytoplasmic organisation being observed subsequently. There was no apparent difference in ALA-PDT response between a multidrug-resistant ovarian carcinoma cell line and its parent line. These results indicate that ALA-PDT has potential for application to therapy of gynaecological malignancies.     

Penetration of oral and topical ciprofloxacin into human aqueous humor

PURPOSE: This study was planned to investigate the penetration of ciprofloxacin into aqueous humor following oral and topical application as a prophylactic antimicrobial agent. METHODS: Forty-six patients undergoing cataract surgery were randomly divided into two groups. In the first group, the patients received 500 mg oral ciprofloxacin eight hours before surgery and in the second, 5 drops of 0.3% ciprofloxacin were applied to the patients every twenty minutes, starting 100 minutes before the surgery. By paracentesis, aqueous samples were taken just before the operation so the interval between the first topical application and paracentesis was 100 minutes. RESULTS: The mean concentration of ciprofloxacin in aqueous humor was 0.63+/-0.29 microg/ml in the first group. The concentration was 0.69+/-0.30 microg/ml in the second group. Both of these mean concentrations were higher than the levels of MIC90 of S.aureus , S. epidermidis, P. aeruginosa and Gram (-) bacteriae. CONCLUSION: As a result, both topically and orally applied ciprofloxacin achieved a significant aqueous concentration. Each route studied might be suitable for surgical prophylaxis or treatment of infections.     

Glycosaminoglycan therapy for long-term diabetic complications?

The tumor of the thoracic cavity, which arose from the ribs, was diagnosed as mesenchymal chondrosarcoma. No distant metastasis was observed. Histologically, the tumor was characterized by the nests of well-defined cartilaginous tissue within a proliferation of primitive mesenchymal cells. Additionally, the deformed blood vessels compressed by the proliferating mesenchymal cells exhibited clear stag-horn appearance. Immunohistochemically, most neoplastic cells that formed multifocal cartilaginous islands were positive for S-100 protein, while the surrounding mesenchymal cells were negative. This is the first report of canine mesenchymal chondrosarcoma of the ribs.     

Delta-aminolevulinic acid-induced fluorescence in normal human lymphocytes

Endogenously generated protoporphyrin IX (PpIX) from exogenous delta-aminolevulinic acid (ALA) has the photodynamic capacity to inactive cancer cells of different origins. The aim of this study was to characterize the ability of normal lymphocytes to transform ALA into PpIX in order to appreciate through further studies changes in pathologic lymphocytes. We investigated in this study PpIX synthesis by normal human lymphocytes using a confocal laser microspectrofluorometer. Live lymphocytes were identified by monoclonal antibody fluorescent labeling. B and T lymphocytes synthesized PpIX (80-100 counts), with a maximum being reached after 4 h ALA incubation. When T subpopulations of lymphocytes were labeled, T4 and T8 changes in fluorescence kinetics were similar, reaching a maximum after 5 h ALA incubation. The influence of monoclonal antibody labeling on this delayed increase for maximum fluorescence is considered. Phytohemagglutinin (PHA, incubation for 72 h) lymphocyte stimulation induced a 100% increase in PpIX fluorescence for T lymphocytes, whereas pokeweed mitogen activation produced an increase of about 50% in the B- or T-lymphocyte signal. Finally, the scanning fluorescence image clearly indicated the inhomogeneity of cytoplasmic ALA-induced PpIX fluorescence, which was probably due to the distribution of mitochondria. The influence of this heterogeneity on PpIX photosensitivity effects is discussed.     

Experimental study of epithelialization of the muscle-only flap in the oral cavity

PURPOSE: The purpose of this study was to observe the epithelialization process of the muscle-only flap used for reconstruction of the oral mucosal defects. MATERIALS AND METHODS:Forty-three male adult Japanese rabbits were used. A superiorly based cleidomastoid muscle flap was designed after vascular assessment. The flap was transferred into the oral cavity to cover a mucoperiosteal defect made in the mandibular alveolus. Epithelialization of the flap was histologically evaluated at designated intervals. RESULTS: The flaps survived without ischemic necrosis. By 8 days postoperation, the flap was infiltrated by acute inflammatory cells and being replaced by granulation tissue originating from the adjacent tissues. The oral epithelial cells advanced onto this granulating muscle flap, with eventual coverage by 21 days. The granulation tissue matured to fibrous tissue with significant contraction by 2 months. At 6 months postoperation, abnormally hyperkeratinized epithelium was seen on the flap. This differed from the surrounding parakeratinized oral epithelium. CONCLUSIONS: The muscle-only flap in the oral cavity epithelializes after the granulation process.     

Nephrogenic rests, nephroblastomatosis, and associated lesions of the kidney

The fetal kidney is formed by the development of nephrons from fetal metanephric blastema surrounding the ureteric bud. The fetal renal tissue matures into normal renal parenchyma during gestation, but, occasionally, fetal tissue persists into infancy as microscopic foci called nephrogenic rests. Nephrogenic rests are found in approximately 1% of infant kidneys at autopsy. Nephrogenic rests are associated with an increased risk of Wilms tumor, and it is theorized that nephrogenic rests undergo neoplastic change into Wilms tumor. Fortunately, this transformation occurs in less than 1% of young children with nephrogenic rests. Nephrogenic rests are associated with many syndromes, including Beckwith-Wiedemann syndrome, hemihypertrophy, and sporadic aniridia. Children with identifiable syndromes, once diagnosed, should be screened for the development of Wilms tumor. Nephrogenic rests are associated with other lesions such as multilocular cystic nephroma and multicystic dysplasia, usually without malignant complications.     

Possible causes of a positive Haemoccult-II test in a population screening study for colorectal neoplasia

In the present paper, common disorders of the oral cavity and oropharynx are described with special emphasis on differential diagnostic aspects. The first part of this presentation covers different inflammatory diseases, mainly focusing on complications like peritonsillar, para- and retropharyngeal abscesses, and Ludwig's angina. These clinical entities can lead to further life-threatening complications, including deep neck infections and mediastinitis. The diagnostic value and necessity of modern imaging in these cases are emphasized. In the second part, the author reports on the incidence, etiology and clinical course of tumors of the oral cavity and oropharynx with special regard to malignancies. Tumors in these areas have been increasing in number over the past decades mainly due to changes in alcohol and nicotine consumption in the developed countries. Diagnostic management includes a thorough clinical evaluation as well as modern imaging for tumor delineation and possible bone infiltration, depending on the site of the original tumor. In addition, therapeutic considerations are discussed, focusing on surgical tumor removal and soft tissue replacement using different pedicled flaps and free flaps. It is also emphasized that postoperative radiotherapy is mandatory in most malignant tumors in this area.