Cryoablation of benign breast tumors: evolution of technique and technology.

We report on improvements in cryoprobe design and techniques of cryoablation as a minimally invasive alternative to open surgery for the treatment of benign breast tumors. In the study, which was conducted in 12 centers, 124 lesions in 102 patients were monitored for a period of 12 months after cryoablation. Two different treatment techniques were used: Double HI FREEZE and Tailored Freeze. In patients treated with the Tailored Freeze technique significantly better results were recorded 12 months after the procedure: the median reduction in tumor volume was 91%, 73% of all tumors treated were nonpalpable, 84% of lesions less than 2.5 cm in maximum diameter were nonpalpable, and none of the 31 mammograms performed yielded abnormal findings. Patient satisfaction was good to excellent in 92% of the patients. The safety profile of this technique was excellent; all complications were minor. Evolution of cryoablation freezing techniques, coupled with improvements in cryoprobe design, has resulted in significant improvements in both safety and effectiveness.

Multicenter trial of sentinel node biopsy for breast cancer using both technetium sulfur colloid and isosulfan blue dye.

OBJECTIVE: To determine the factors associated with false-negative results on sentinel node biopsy and sentinel node localization (identification rate) in patients with breast cancer enrolled in a multicenter trial using a combination technique of isosulfan blue with technetium sulfur colloid (Tc99). SUMMARY BACKGROUND DATA: Sentinel node biopsy is a diagnostic test used to detect breast cancer metastases. To test the reliability of this method, a complete lymph node dissection must be performed to determine the false-negative rate. Single-institution series have reported excellent results, although one multicenter trial reported a false-negative rate as high as 29% using radioisotope alone. A multicenter trial was initiated to test combined use of Tc99 and isosulfan blue. METHODS: Investigators (both private-practice and academic surgeons) were recruited after attending a course on the technique of sentinel node biopsy. No investigator participated in a learning trial before entering patients. Tc99 and isosulfan blue were injected into the peritumoral region. RESULTS: Five hundred twenty-nine patients underwent 535 sentinel node biopsy procedures for an overall identification rate in finding a sentinel node of 87% and a false-negative rate of 13%. The identification rate increased and the false-negative rate decreased to 90% and 4.3%, respectively, after investigators had performed more than 30 cases. Univariate analysis of tumor showed the poorest success rate with older patients and inexperienced surgeons. Multivariate analysis identified both age and experience as independent predictors of failure. However, with older patients, inexperienced surgeons, and patients with five or more metastatic axillary nodes, the false-negative rate was consistently greater. CONCLUSIONS: This multicenter trial, from both private practice and academic institutions, is an excellent indicator of the general utility of sentinel node biopsy. It establishes the factors that play an important role (patient age, surgical experience, tumor location) and those that are irrelevant (prior surgery, tumor size, Tc99 timing). This widens the applicability of the technique and identifies factors that require further investigation.

Roles for zebrafish focal adhesion kinase in notochord and somite morphogenesis.

We have cloned zebrafish focal adhesion kinase (Fak) and analyzed its subcellular localization. Fak protein is localized at the cortex of notochord cells and at the notochord-somite boundary. During somitogenesis, Fak protein becomes concentrated at the basal region of epithelial cells at intersomitic boundaries. Phosphorylated Fak protein is seen at both the notochord-somite boundary and intersomitic boundaries, consistent with a role for Fak in boundary formation and maintenance. The localization of Fak protein to the basal region of epithelial cells in knypek;trilobite double mutant embryos shows that polarization of Fak distribution in the somite border cells is independent of internal mesenchymal cells. In addition, we show that neither Notch signaling through Suppressor of Hairless (SuH) nor deltaD is necessary for the wild-type segmental pattern of fak mRNA expression in the anterior paraxial mesoderm. However, nonsegmental expression of fak mRNA occurs with ectopic activation of Notch signaling through SuH and also in fused somite and beamter mutant embryos, indicating that there are multiple regulators of fak mRNA expression. Our results suggest that Fak plays a central role in notochord and somite morphogenesis.

Somites in zebrafish doubly mutant for knypek and trilobite form without internal mesenchymal cells or compaction.

In vertebrates, paraxial mesoderm is partitioned into repeating units called somites. It is thought that the mechanical forces arising from compaction of the presumptive internal cells of prospective somites cause them to detach from the unsegmented presomitic mesoderm [1-3]. To determine how prospective somites physically segregate from each other, we used time-lapse microscopy to analyze the mechanics underlying early somitogenesis in wild-type zebrafish and in the mutants trilobite(m209) (tri), knypek(m119) (kny), and kny;tri, which are defective in convergent extension during gastrulation. Formation of somite boundaries in all of these embryos involved segregation, local alignment, and cell-shape changes of presumptive epitheloid border cells along nascent intersomitic boundaries. Although kny;tri somites formed without convergence of the presomitic mesoderm and were composed of only two cells in their anteroposterior (AP) dimension, they still exhibited AP intrasegmental polarity. Furthermore, morphogenesis of somite boundaries in these embryos proceeded in a manner similar to that in wild-type embryos. Thus, intersomitic boundary formation in zebrafish involves short-range movements of presumptive border cells that do not require mechanical forces generated by internal cells or compaction of the presomitic mesoderm.

Confocal microscopic analysis of morphogenetic movements.

Confocal microscopy is an excellent means of imaging cellular dynamics within living zebrafish embryos because it provides a means of optically sectioning tissues that have been labeled with specific fluorescent probe molecules. In order to study genetically encoded patterns of cell behavior that are involved in the formation of germ layers and various organ primordia, it is possible to vitally stain an entire zebrafish embryo with one or more fluorescent probe molecules and then examine morphogenetic behaviors within specific cell populations of interest using time-lapse confocal microscopy. There are two major advantages to this "bulk-labeling" approach: (1) the applied fluorescent probe (a contrast-enhancing agent) allows all of the cells within an intact zebrafish embryo to be rapidly stained; (2) the morphogenetic movements and shape changes of hundreds of cells can then be examined simultaneously in vivo using time-lapse confocal microscopy. The neutral fluorophore Bodipy 505/515 and its sphingolipid-derivative Bodipy-C5-ceramide are particularly useful, nonteratogenic vital stains for imaging cellular dynamics in living zebrafish embryos. These photostable fluorescent probes (when applied with 2% DMSO) percolate through the enveloping layer epithelium of the embryo, and localize in yolk-containing cytoplasm and interstitial space, respectively, owing to their different physiochemical characteristics. Bodipy-ceramide, for instance, remains highly localized to interstitial fluid once it accumulates within a zebrafish embryo, allowing the boundaries of deep cells to be clearly discerned throughout the entire embryo. Through the use of either of these fluorescent vital stains, it is possible to rapidly convert a developing zebrafish embryo into a strongly fluorescent specimen that is ideally suited for time-lapse confocal imaging. For zebrafish embryos whose deep cells have been intentionally "scatter-labeled" with fluorescent lineage tracers (e.g., fluorescent dextrans), sequential confocal z-series (i.e., focus-throughs) of the embryo can be rendered into uniquely informative 3D time-lapse movies using readily available image-processing programs. Similar time-lapse imaging, combined with rapidly advancing computer-assisted visualization techniques, may soon be applied to study the dynamics of GFP-fusion proteins in vivo, as well as other types of synthetic probe molecules designed to reveal the cytological processes associated with the patterning and morphological transformations of the zebrafish's embryonic tissues.

Educating residents for managed care: report on a multidisciplinary conference.

A growing number of residency programs are preparing their graduates for the realities of managed care practice. In 1996, The Cleveland Clinic Foundation, a private, nonprofit academic medical center, hosted a two-day conference on managed care education to develop innovative instructional and evaluative approaches that, where appropriate, would build on existing expertise. The conference was attended by invited national experts who had a stake in residents' education: clinical faculty, residents, medical educators, executives of managed care organizations, and representatives of other interested organizations. Participants spent much of their time in four small break out groups, each focusing on one of the following topics that were judged particularly relevant to managed care: preventive and population-based medicine, appropriate utilization of resources, clinician-patient communication, and interdisciplinary team practice. Participants shared existing materials, discussed teaching goals and objectives, and generated ideas for teaching methods, teaching materials, and evaluative methods for their respective topics. The authors summarize the recommendations from the four groups, with an overview of the issues that emerged during the conference concerning curriculum development, integration of managed care topics into existing curricula, staging of the curriculum, experiential teaching methods, negative attitudes and resistance, evaluation of trainees and profiling, program assessment, faculty development, and cooperation between academic medical centers and managed care organizations.

Phototoxicity of argon laser irradiation on biofilms of Porphyromonas and Prevotella species.

Species of Prevotella (Pr.) and Porphyromonas (Po.) and other microorganisms were cultivated as biofilms on agar medium and examined for their susceptibility to argon laser irradiation (continuous mode; wavelengths, 488-514 nm; fluences, 20-200 J cm(-2)). Fluences of 35 to 80 J cm(-2) inhibited biofilm growth in Po. endodontalis, Po. gingivalis, Pr. denticola, Pr. intermedia, Pr. melaninogenica and Pr. nigrescens. A fluence of 70 J cm(-2) did not affect biofilm growth in species of Bacillus, Candida, Enterobacter, Proteus, Pseudomonas, Staphylococcus and Streptococcus. The phototoxic effects of argon laser irradiation against Prevotella and Porphyromonas species were: (1) caused by the radiation alone; (2) modified by biofilm age; (3) dependent on the presence of atmospheric oxygen; (4) influenced by medium supplements of hemin, hemoglobin and blood; (5) greater when compared with other microbial species; (6) demonstrated without augmentation with an exogenous photosensitizer; and (7) apparently unrelated to the protoporphyrin content of the cells. Overall, these in vitro findings suggest that low doses of argon laser radiation may be effective in the treatment and/or prevention of clinical infections caused by biofilm-associated species of Prevotella or Porphyromonas.

Measurement and manipulation of cytosolic pH in polarizing zygotes.

The role of cytosolic pH (pHc) in establishment and expression of developmental polarity was examined in zygotes of the brown alga Pelvetia. pHc was measured and manipulated at specific developmental stages during the first zygotic cell cycle. pHc was measured using pH-sensitive microelectrodes and by confocal ratio imaging of dextran-conjugated SNARF 1 (dc SNARF 1) loaded cells. The two techniques yielded very similar values of pHc in the cellular cortex, but ratio imaging was not effective in measuring endoplasmic pHc values. As zygotes formed a developmental axis, cortical pHc decreased abruptly by approximately 0.1 units, and a small but significant difference in pHc was detected at the thallus and rhizoid poles. The cortical cytosol was relatively acidic at the presumptive rhizoid pole. The magnitude of the pHc difference increased following initiation of rhizoid growth. pHc was manipulated by treating zygotes with membrane-permeant weak acids (propionic and benzoic acid) or bases (methylamine and procaine), which effectively clamp pHc to specific values in a concentration-dependent manner. pHc values in treated zygotes were measured for each concentration of acid or base, and a dose response curve was generated. Zygotes in which pHc had been clamped were examined for their ability to form a developmental axis and to initiate rhizoid outgrowth (germination). Both developmental processes were inhibited by relatively small (0.2-0.3 pH units) perturbations of pHc. The permissive ranges of pHc were slightly different, germination (permissive pHc range-pH 7.0 to 7.7) being more acid tolerant than axis formation (permissive pHc range-7.2 to 7.8).

Sensitivity of Porphyromonas and Prevotella species in liquid media to argon laser.

The phototoxicity of argon laser irradiation was studied in aqueous suspensions of Porphyromonas endodontalis (American Type Culture Collection [ATCC] 35406), Porphyromonas gingivalis (ATCC 33277), Prevotella denticola (ATCC 33184) and two strains of Prevotella intermedia (ATCC 15033 and 49046), all "black-pigmented bacteria," BPB, that accumulate cellular porphyrins. Several of these species have been implicated in the etiology of periodontal disease. Non-black-pigmented bacteria were also studied to test the specificity of irradiation as a potential photodynamic treatment for periodontal infections. Cell suspensions were irradiated with an argon laser at fluences of 20-200 J/cm2. When cultured in hemin-supplemented media, ATCC 15033 was the most sensitive to irradiation. However, a second strain of the same species (ATCC 49046) was resistant. The photosensitivity of other species ranked ATCC 33277 > 35406 = 33184 = 35496. When hemin was replaced in media by hemoglobin, ATCC 33277 became resistant to irradiation. Protoporphyrin IX content in BPB cells was shown not to be a major factor determining photosensitivity. Oxygen was required during irradiation for BPB species to be affected. Non-black-pigmented bacteria were much less sensitive to irradiation than BPB.

Aiding the novice practitioner in contact lens selection.

A comprehensive study was performed to assist the new graduate and novice contact lens practitioner in the choice of the optimal lens for each case scenario, as well as provide guidance for inventory selection. The survey was distributed to the clinical staff at the State University of New York, State College of Optometry, in order to take advantage of the knowledge of the experienced contact lens fitter. There appear to be clinically significant relationships between lens characteristics and performance ratings.

Serum glucose: effects on tumor and normal tissue accumulation of 2-[F-18]-fluoro-2-deoxy-D-glucose in rodents with mammary carcinoma.

The positron-emitter-labeled glucose analogue 2-[fluorine-18]-fluoro-2-deoxy-D-glucose (FDG) accumulates into many cancers after intravenous injection, but the effect of serum glucose levels on FDG uptake in the tumor has not been extensively studied. In vitro, elevated media glucose levels markedly diminished FDG and FDG 6-phosphate uptake and retention in human adenocarcinoma cells, while insulin had no effect. Mammary cancers were established subcutaneously in 12 rats. Six control rats with mammary tumors were fasted overnight. Hyperglycemia was established in six rats by means of continuous glucose infusion (glucose clamp). All animals were then intravenously administered 50 microCi of FDG. Serum glucose levels were 87 mg/dL (4.83 mmol/L) in the control animals and more than 900 mg/dL (49.9 mmol/L) in the hyperglycemic animals. One hour after injection of FDG, mean F-18 uptake in the tumor, brain, small bowel, and ovaries was 2.7-9.7 times lower in the hyperglycemic animals (P less than .02). Mean F-18 activity in the kidneys tended to be somewhat higher in the hyperglycemic animals. FDG uptake in other tissues was comparable between the control and hyperglycemic groups. These data suggest that high serum glucose levels may substantially impair visceral tumor imaging with FDG positron emission tomography.

Antibacterial activity of dental implant metals.

This study was conducted to investigate the specific in vitro antibacterial effect of seven dental implant metals on oral bacteria which have often been identified in failing implants. The metals chosen for evaluation were titanium, chromium, cobalt, aluminum, iron, gold, and vanadium. These metals were selected because they are contained in many commonly used dental implants. The bacteria selected for this study included isolates of Porphyromonas endodontalis, Porphyromonas gingivalis, Prevotella intermedia, Prevotella melaniogenica, Actinobacillus actinomycetemcomitans, Actinomyces naeslundii, and Actinomyces viscosus. Sets of tubes containing either supplemented trypticase soy broth, brain-heart infusion-yeast extract, or brain-heart infusion-yeast extract with 5 percent defibrinated rabbit blood were aseptically prepared with doubling dilutions of the seven metals starting at an initial concentration of 500 micrograms/ml and terminating at 0 microgram/ml. Cultures of each organism were inoculated into each set of broth tubes containing all concentrations of metals. Tubes were incubated either anaerobically or in an atmosphere of 5 percent carbon dioxide for 48 hours at 35 degrees C and then assayed for ATP content which was proportional to the viable cellular biomass. The results showed that, although being bacteria and concentration dependent, all seven metals suppressed the growth of each organism. The rank order of antibacterial activity expressed by dental implant metals was gold > titanium > cobalt > vanadium > aluminum > chromium > iron.

Improved monoclonal antibody tumor/background ratios with exchange transfusions.

Blood exchange transfusions were performed in nude rats with subcutaneous HTB77 human ovarian carcinoma xenografts in an attempt to improve specific monoclonal antibody (MoAb) tumor/non-tumor uptake ratios. Animals were injected intravenously with both 131I-5G6.4 specific and 125I-UPC-10 non-specific MoAb. Twenty-four hours later 65-80% of the original blood was exchanged with normal heparinized rat blood and then these rodents were sacrificed. Exchange transfusion significantly (P less than 0.05) decreased normal tissue activities of 131I (except for muscle) by 63-85%, while tumor activity decreased only 5%. Tumor to background ratios increased from 0.1-0.8 to 2.3-6.3. Exchange transfusions substantially enhance tumor/normal tissue antibody uptake ratios and, along with plasmapheresis, may be useful in enhancing antibody localization in vivo, particularly for therapy.

Determinants of acute hemodynamic and electrophysiologic effects of verapamil in humans: role of myocardial drug uptake.

To examine the relationship between myocardial verapamil content (MVC) and acute effects in humans, coronary sinus catheterization was used in 22 patients to determine myocardial uptake of verapamil after bolus intravenous (i.v.) verapamil (4 mg) injection. Verapamil-induced effects on hemodynamic and electrophysiologic parameters were measured simultaneously and correlated with MVC per unit baseline coronary sinus blood flow (MVC:F). Myocardial uptake of verapamil was rapid: peak MVC (1.2 +/- 0.2% of injected dose) occurred at 5.4 +/- 0.4 min; at 30 min, residual MVC was 71.1 +/- 3.4% of maximum. Peak MVC:F in individual patients was inversely related to the extent of coronary artery disease (p less than 0.005) but not to left ventricular (LV) systolic function. Verapamil produced significant (p less than 0.001) early reductions in arterial pressure and systemic vascular resistance (SVR); cardiac index (CI) increased, left ventricular (LV) positive dP/dt was unchanged. Verapamil prolonged (p less than 0.01) PR and AH intervals (maximum at 12-18 min) and atrioventricular (AV) nodal effective and functional refractory periods (ERP, FRP) (maximum at 30 min). In individual patients, the extent of changes in AH intervals (r = 0.69; p less than 0.05) and LV dP/dt (r = 0.62; p less than 0.05) correlated with peak MVC:F. We conclude that after i.v. injection, verapamil uptake by the human myocardium is rapid and more extensive in patients with minor coronary artery disease. Despite the hysteresis between MVC and drug effects, MCV is a determinant of inotropic and electrophysiologic effects of verapamil.

Comparison of the antimicrobial effectiveness of regular and fresh scent Clorox.

The antimicrobial properties of two different compositions of sodium hypochlorite were compared in a tube dilution study. Absorbent paper points were contaminated with Streptococcus faecalis or Candida albicans and exposed to 5.25% or 2.62% concentrations of "regular" or "fresh scent" sodium hypochlorite (Clorox) for periods ranging from 15 to 120 s. The points were then removed from the sodium hypochlorite solution, placed into a growth medium, incubated, and the presence or absence of growth recorded. Results showed that formulary changes involved in the manufacture of the "fresh scent" sodium hypochlorite had no apparent effect on its antimicrobial properties, as both compositions proved equally effective against the test organisms at each concentration evaluated.

Bactericidal effects of human neutrophils and sera on selected endodontic pathogenic bacteria in an anaerobic environment.

Phagocytosis by normal human neutrophils and the bactericidal activity of normal human pooled serum were measured under anaerobic conditions on six endodontic pathogenic bacterial species from the genera Actinomyces, Bacteroides, Fusobacterium, Peptostreptococcus and Streptococcus. The results revealed that Actinomyces viscosus was not killed either by human neutrophils or by 20 per cent serum; Peptostreptococcus anaerobius was not killed by either 20 per cent or 95 per cent serum; and Bacteroides intermedius was susceptible to both 20 per cent and 95 per cent serum, but was not killed by neutrophils after exposure for 20 minutes. The other species showed intermediate susceptibility patterns. Such results suggest that oral pathogens vary with regard to the bactericidal effects of these two host defense mechanisms, and that this variation may at least in part explain the relative quantities of microbes recovered in certain oral infections.