Longitudinal, quantitative monitoring of therapeutic response in 3D in vitro tumor models with OCT for high-content therapeutic screening.

In vitro three-dimensional models of cancer have the ability to recapitulate many features of tumors found in vivo, including cell-cell and cell-matrix interactions, microenvironments that become hypoxic and acidic, and other barriers to effective therapy. These model tumors can be large, highly complex, heterogeneous, and undergo time-dependent growth and treatment response processes that are difficult to track and quantify using standard imaging tools. Optical coherence tomography is an optical ranging technique that is ideally suited for visualizing, monitoring, and quantifying the growth and treatment response dynamics occurring in these informative model systems. By optimizing both optical coherence tomography and 3D culture systems, it is possible to continuously and non-perturbatively monitor advanced in vitro models without the use of labels over the course of hours and days. In this chapter, we describe approaches and methods for creating and carrying out quantitative therapeutic screens with in vitro 3D cultures using optical coherence tomography to gain insights into therapeutic mechanisms and build more effective treatment regimens.

Chemically specific imaging of cryptosporidium oocysts using coherent anti-Stokes Raman scattering (CARS) microscopy.

We demonstrate the application of coherent anti-Stokes Raman scattering microscopy for the rapid, label-free chemical imaging of waterborne pathogens. Chemically selective images of cryptosporidium were acquired in just a few seconds using coherent anti-Stokes Raman scattering microscopy, demonstrating its capability for the rapid detection of cryptosporidium at the single oocyst level. We discuss the applicability of such a technique in a near-real time automated water testing system.

Adaptive optics for enhanced signal in CARS microscopy.

We report the use of adaptive optics with coherent anti-Stokes Raman scattering (CARS) microscopy for label-free deep tissue imaging based on molecular vibrational spectroscopy. The setup employs a deformable membrane mirror and a random search optimization algorithm to improve signal intensity and image quality at large sample depths. We demonstrate the ability to correct for both system and sample-induced aberrations in test samples as well as in muscle tissue in order to enhance the CARS signal. The combined system and sample-induced aberration correction increased the signal by an average factor of approximately 3x for the test samples at a depth of 700 microm and approximately 6x for muscle tissue at a depth of 260 microm. The enhanced signal and higher penetration depth offered by adaptive optics will augment CARS microscopy as an in vivo and in situ biomedical imaging modality.

A truncated precursor form of prostate-specific antigen is a more specific serum marker of prostate cancer.

Prostate-specific antigen (PSA) is a widely used serum marker for prostate cancer (PCa) but has limited specificity for distinguishing early PCa from benign prostatic hyperplasia, because both PCa and benign prostatic hyperplasia release PSA into the serum. We have identified previously a truncated form of precursor PSA (pPSA) in prostate tumor extracts consisting of PSA with a serine-arginine pro leader peptide ([-2]pPSA) instead of the normally expressed 7 amino acid pro leader peptide. In the current study we developed monoclonal antibodies to detect [-2]pPSA and other isoforms of pPSA for Western blot analysis. PSA was immunoaffinity purified from 100 to 200 ml of serum from each of five men with biopsy-proven cancer and three biopsy-negative men, all with total PSA levels in the diagnostically relevant range near 10 ng/ml. The truncated [-2]pPSA was estimated to range from 25 to 95% of the free PSA in the five PCa samples but only 6-19% of the free PSA in the biopsy-negative men. Immunohistochemical studies showed positive staining for [-2]pPSA in PCa epithelium and that [-2]pPSA was enriched in cancer cell secretions. In vitro activation studies showed that human kallikrein 2 and trypsin readily activated full-length pPSA but were unable to activate [-2]pPSA to mature PSA. Thus, [-2]pPSA, once formed, is a stable but inactive isoform of PSA. Truncated [-2]pPSA may represent an important new diagnostic marker for the early detection of PCa.

Development of a dual monoclonal antibody immunoassay for total human kallikrein 2.

BACKGROUND: Human kallikrein 2 (hK2) shares 80% sequence identity with prostate-specific antigen (PSA). Because both hK2 and hK2-alpha(1)-antichymotrypsin (hK2-ACT) complexes have been identified in patient sera, we devised an immunoassay for total hK2 [(thK2); hK2 and hK2-ACT] and evaluated it in healthy subjects and patients with prostate disease. METHODS: We developed monoclonal antibodies (mAbs) with high specificity for hK2 and hK2-ACT and minimal cross-reactivity to PSA. Using these mAbs, a sandwich assay was developed and its specificity for forms of hK2 was assessed. Serum samples (n = 1035) from healthy volunteers, patients with increased PSA, and men who had undergone radical prostatectomy were assayed for thK2. We also measured thK2 in samples before and after storage under common laboratory conditions. RESULTS: The minimum detectable concentration in the thK2 assay was 0.008 microg/L, and PSA cross-reactivity was <0.001%. The assay detected prohK2 and three different hK2-serum protease complexes. The median serum concentration of thK2 in control samples (0.013 microg/L) was significantly lower than the median in samples from patients with increased PSA concentrations (0.085 microg/L). Immunoreactive hK2 changed little in samples stored for up to 1 month at -70 degrees C. CONCLUSIONS: The thK2 assay recognizes all forms of hK2 that have been found in bodily fluids to date.

A single gene (yes) controls pigmentation of eyes and scales in Heliothis virescens.

A yellow-eyed mutant was discovered in a strain of Heliothis virescens, the tobacco budworm, that already exhibited a mutation for yellow scale, y. We investigated the inheritance of these visible mutations as candidate markers for transgenesis. Yellow eye was controlled by a single, recessive, autosomal factor, the same type of inheritance previously known for y. Presence of the recombinant mutants with yellow scales and wild type eyes in test crosses indicated independent segregation of genes for these traits. The recombinant class with wild type scales and yellow eyes was completely absent and there was a corresponding increase of the double mutant parental class having yellow scales and yellow eyes. These results indicated that a single factor for yellow eye also controlled yellow scales independently of y. This gene was named yes, for yellow eye and scale. We hypothesize that yes controls both eye and scale color through a deficiency in transport of pigment precursors in both the ommochrome and melanin pathways. The unlinked gene y likely controls an enzyme affecting the melanin pathway only. Both y and yes segregated independently of AceIn, acetylcholinesterase insensitivity, and sodium channel hscp, which are genes related to insecticide resistance.

Is auditory imagery defective in patients with auditory hallucinations?

BACKGROUND: A variant of the 'inner speech' theory of auditory verbal hallucinations in schizophrenia suggests that there is an abnormality of the relationship between the 'inner voice' and 'inner ear', such that hallucinators are unable to distinguish inner 'imagined' speech from real external speech, and so misrecognize inner speech as alien. METHODS: Five experiments were carried out comparing 12 schizophrenic patients who were highly prone to hallucinate, with seven patients who were not, on a series of auditory imagery tasks that are differentially dependent on inner voice/inner ear partnership for successful performance: parsing meaningful letter/number strings; the verbal transformation effect; phoneme judgements; pitch judgements, and homophony and rhyme judgements. RESULTS: Contrary to our hypothesis, there was no evidence that the group with the propensity to hallucinate were impaired on tasks requiring normal inner ear/inner voice partnership. CONCLUSIONS: Together with previous work indicating no impairment of the phonological loop in patients who hallucinate, these results suggest that inner speech and auditory verbal hallucinations are not connected in a simplistic or direct way. Indeed, a reappraisal of psychological models of hallucinations in general may be warranted.

Tricyclic antidepressants in adolescent depression. A case report.

A boy aged 11 years 11 months, with normal premorbid personality, presented with a severe depressive episode with somatic and psychotic features. A clinical response to amitriptyline was complicated by ECG changes leading to the abrupt withdrawal of amitriptyline, with the development of a withdrawal syndrome. Further trials of antidepressant medication were unsuccessful, including paroxetine (clinical deterioration), lofepramine (ECG changes and clinical deterioration), and trazodone (priapism). Finally, a good clinical response to dothiepin augmented with lithium was achieved. ECG changes were assessed as being idiosyncratic responses to medication, rather than ischaemic in nature. A dose/response relationship with dothiepin was observed. All medication was successfully stopped after 26 months of treatment. Clinical phenomena relevant to the development of guidelines for use of tricyclic antidepressants in childhood and adolescence are discussed.

Development of monoclonal antibodies specific for human glandular kallikrein (hK2): development of a dual antibody immunoassay for hK2 with negligible prostate-specific antigen cross-reactivity.

OBJECTIVES: Human glandular kallikrein (hK2) is a protein that is 80% homologous to prostate-specific antigen (PSA), and, like PSA, is localized to the prostate. We developed a specific immunoassay for hK2 that can be used to evaluate its clinical diagnostic utility. METHODS: We developed monoclonal antibodies (mAbs) specific for hK2 by immunizing with hK2 and screening for clones reactive with hK2 and not PSA. Prototype sandwich assays using these mAbs were tested, and the optimum pair selected. Purified hK2 was used as standard and PSA cross-reactivity was assessed in the assay. Both hK2 and hK2-alpha1-antichymotrypsin (ACT) complexes have been identified in sera of patients with prostate cancer (PCa). Serum samples (n = 671) from healthy volunteers and patients with prostate disease were assayed for hK2 and PSA levels. RESULTS: The assay had a detection limit of less than 0.12 ng/mL and a less than 0.5% cross-reactivity with PSA. The assay preferentially detected free hK2 with a 3.5-fold higher molar response than with hK2-ACT. The mean serum concentration of hK2 in normal control samples was low (0.33 and 0.37 ng/mL for normal healthy men and women, respectively) but was elevated in patients with prostate disease (0.86 and 6.77 ng/mL for patients with benign prostatic hyperplasia and PCa, respectively). Negligible cross-reactivity to hK2 was measured by Tandem PSA assays (Hybritech). CONCLUSIONS: Significant concentrations of hK2, relative to PSA, were detected in human serum, especially in patients with prostate disease. Serum hK2 concentrations were not proportional to PSA concentration. Therefore, hK2 has the potential to be an independent and clinically useful marker for PCa.

Molecular forms of prostate-specific antigen and human kallikrein 2 (hK2) in urine are not clinically useful for early detection and staging of prostate cancer.

OBJECTIVES: Prostate-specific antigen (PSA), a member of the human kallikrein (hK) family, is the most important tumor marker for early detection, staging, and monitoring of men with prostate cancer today. However, the sensitivity of serum PSA is not sufficient to be used alone for prostate cancer screening. Recently, it was reported that the serum-to-urinary total PSA ratio improves the detection of men with prostate cancer, especially in men with a serum total PSA level between 4.0 and 10.0 ng/mL. We tested this hypothesis by evaluating the clinical usefulness of this PSA ratio as well as the use of the different molecular forms of PSA and human kallikrein 2 (hK2) in urine for detection and staging of prostate cancer. METHODS: One hundred ten fresh, midstream urine specimens (prostate cancer 62, benign prostatic hyperplasia [BPH] 38, healthy male control 5, women 5) were collected. Serum total PSA, urine total PSA, urinary free PSA, urinary alpha 1-antichymotrypsin-bound PSA, and urinary hK2 levels were determined by monoclonal antibody assays (Hybritech Inc.). The serum-to-urinary total PSA ratio was calculated. RESULTS: The serum-to-urinary total PSA ratio did not accurately distinguish between men with BPH and men with prostate cancer. There was no significant difference between the urinary levels of any of the molecular forms of PSA or hK2 between men with prostate cancer and men with BPH. Among men with prostate cancer, neither urinary hK2 nor urinary levels of any of the molecular forms of PSA correlated with age, pathologic stage, or Gleason grade. CONCLUSIONS: In our study, the serum-to-urinary total PSA ratio did not improve the detection of men with prostate cancer. Furthermore, measurement of the molecular forms of PSA and hK2 in urine did not improve the detection or staging of prostate cancer over serum PSA alone.

On-line monitoring of powder blend homogeneity by near-infrared spectroscopy.

Near-infrared spectroscopy is evaluated as an on-line technique for monitoring the homogeneity of a pharmaceutical blend during the blending process. Blends containing 10% sodium benzoate (model active), which provided an aromatic functionality typical of many pharmaceutical compounds, 39% microcrystalline cellulose (Avicel PH102), 50% lactose, and 1% magnesium stearate were developed to mimic the properties of an actual pharmaceutical blend. A twin-shell V-blender was modified to allow installation of a diffuse reflectance fiber-optic probe at the axis of rotation, and spectra were collected during three experiments using a commercially available near-infrared spectrophotometer. In each experiment, blender control and spectral data collection were controlled by a compilation of software packages. The experiments detected spectral changes which eventually converged to a point of constant variance. Further analysis of the spectral data showed the blend is homogeneous long before a typical blending period is complete. Near-infrared spectroscopy has proven to be a feasible and effective method for the "real time" noninvasive determination of homogeneity in a pharmaceutical blend.

Rumination in adults: two case histories.

Rumination has been reported to be a relatively rare disorder of eating during infancy. Over the past decade, there appears to be a renewed interest in and recognition of adult rumination. Although some authors believe adult rumination is benign, others have begun to link it with both eating disorders and depressive symptoms. This paper presents two adult cases whose rumination was associated with anorexia and bulimia nervosa. More identification and study of adult rumination is needed to clarify its course and medical significance.

Investigation of methods to detect mechanically recovered meat in meat products - III: Microscopy.

This was a preliminary study to investigate whether hyaline cartilage could be easily identified in mechanically recovered meat (MRM) and whether its presence could be used as a possible marker for MRM in meat products. MRMs produced from beef, pork, lamb, chicken and turkey, using a variety of machine types and processing conditions, were compared to both minced and colloid milled hand-deboned samples, using a chemical staining technique followed by examination using the light microscope. The methodology was tested on various mixtures of MRM and hand-deboned meat. Although this technique, as with most microscopy techniques generally, is not suitable for quantitative determinations, the results indicate that light microscopy could be used as a useful screening method.

Physician pregnancy: male and female colleagues' attitudes.

A survey questionnaire was mailed to residents and faculty at a midwestern medical school to assess male and female attitudes toward colleagues' pregnancies. A total of 67% (N = 97) of the 145 faculty and 48% (N = 103) of the 214 residents completed surveys, yielding an overall return rate of 56% (N = 200). Among faculty, responses on only 1 of the 15 items showed a significant difference by gender. Residents' responses, however, showed statistically significant gender differences on 8 of the 15 items. More female than male residents felt that pregnant physicians maintain job performance and interest in medicine. More male than female residents believed pregnancy was disruptive to relationships with colleagues and viewed women of childbearing age as a risk to the optimum functioning of a department. The authors discuss reasons for the gender differences in attitude found among the residents and suggest possible interventions.