Expression and localization of inducible and endothelial nitric oxide synthase in the rat ovary. Effects of gonadotropin stimulation in vivo.

Nitric oxide is reportedly involved in the regulation of several ovarian processes, yet the isoforms of nitric oxide synthase (NOS) expressed in the ovary are unknown. Our purpose was to identify and localize NOS isoenzymes in the rat ovary and to examine++ if mRNA expression of NOS isoenzymes change after gonadotropin stimulation. Using reverse transcriptase-PCR, we demonstrated that inducible (iNOS) and endothelial (eNOS), but not neuronal, NOS mRNAs are expressed in the ovary. In a gonadotropin-stimulated rat model, unstimulated ovaries had the highest levels of iNOS mRNA as quantified by ribonuclease protection assay. After gonadotropin injection, iNOS mRNA declined to undetectable levels in ovaries containing ovulatory follicles before increasing slighty in ovaries containing copora lutea. In situ hybridization studies localized iNOS to granulosa cells of secondary follicles and small antral follicles. Western blots of unstimulated ovaries demonstrated iNOS protein. In contrast to iNOS, eNOS mRNA levels, determined by quantitative PCR, increased after gonadotropin stimulation and peaked in ovaries containing ovulatory follicles before declining in the luteal phase. eNOS protein was localized to blood vessels in the ovary by immunohistochemistry. We conclude that two isoforms of NOS are expressed in the ovary and the mRNA levels for these isozymes are differentially regulated.

Modulation of inducible nitric oxide synthase expression in astroglial cells.

Nitric oxide is produced in the CNS by both constitutive and inducible isoforms of nitric oxide synthase. Once nitric oxide synthase is transcriptionally induced in astrocytes in vitro, these cells release large amounts of nitric oxide tonically. Glial cell-derived nitric oxide can be toxic to neurons and oligodendrocytes and is implicated in a variety of neuropathologies, suggesting that the expression of nitric oxide synthase in glia must be finely regulated. From northern and western blot analysis we have identified various agents (transforming growth factor-beta, nitric oxide, receptor agonists) that modulate cytokine-induced expression of nitric oxide synthase mRNA in astrocytes. This suggests that the magnitude and duration of nitric oxide production from activated astrocytes in vivo may be determined by signals from adjacent neurons and microglial cells.

Nitric oxide synthase type II expression by different cell types in MHV-JHM encephalitis suggests distinct roles for nitric oxide in acute versus persistent virus infection.

Intranasal inoculation with mouse hepatitis virus strain JHM (MHV-JHM) results in acute meningoencephalitis. We found NOS II mRNA expression in brains of acutely infected animals on days 5 through 7 after infection. In situ hybridization and immunohistochemistry demonstrated NOS II message and protein in infiltrating macrophages. Persistent infection with MHV-JHM results in chronic demyelinating encephalomyelitis. NOS II mRNA was detected in persistently infected spinal cords. In situ hybridization and immunohistochemistry showed expression of NOS II in astrocytes in and around demyelinated lesions. These results suggest the role of NO release in acute versus persistent infection with this virus, and its contribution to the resulting pathology, may be different.

Expression of nitric oxide synthase type II in the spinal cord under conditions producing thermal hyperalgesia.

There is evidence supporting spinal cord nitric oxide (NO) production in the mechanisms underlying hyperalgesia, presumed to arise from the activity of neuronal nitric oxide synthase type I (NOS I). Intrathecal administration of interleukin-1 beta and interferon-gamma to rats results in a thermal hyperalgesia which peaks at 2 h post-injection but which is undetectable 8 h post-injection. Expression of mRNA for nitric oxide synthase type II (NOS II) was detected by reverse transcription-polymerase chain reaction followed by Southern hybridization utilizing specific oligonucleotides in spinal cord tissue from animals 4 h and 8 h after cytokine injection, but not at longer time points. NOS II protein was detected in soluble fractions of spinal cords from animals 4 h and 8 h after cytokine injection. In situ hybridization for NOS II mRNA revealed positive cells bilaterally in the spinal cord 4 h after cytokine injection in a perivascular distribution and scattered throughout the gray and white matter. Immunohistochemistry for NOS II showed a similar distribution which could only be partially accounted for by macrophages/microglia. These results provide evidence for induction of NOS II expression under conditions producing thermal hyperalgesia and suggest a possible role in this behavior for the production of NO by a variety of cell types in the CNS.

The usefulness of pathologists' assistants.

The usefulness of pathologists' assistants (PAs) has not been assessed rigorously. Data from a time-motion self-report log generated by an Allegheny General Hospital (Pittsburgh, PA) PA and from the corresponding surgical specimen logs were reviewed to determine the daily distribution of PA time and multiple parameters of practice for gross examination of specimens. Using these data in specific scenarios, PA and non-PA practices were compared. The majority of the PA's time (56.5%) was spent performing gross examination of surgical biopsy specimens. The average cost of gross examination per specimen for a PA and a pathologist was $4.37 and $15.19, respectively. In this practice setting, $91,970.00 is saved per year by the use of a PA. The use of PAs instead of pathologists results in considerable practice cost savings ($560,000 in a practice of 50,000 specimens) or saves pathologists time to perform other necessary functions. PAs are highly useful in an era of cost containment.

Pathologists' assistants practice: a measurement of performance.

Despite their widespread utilization, little is known about the quality of pathologists' assistants' services. Pathologists' assistants' performance was compared with pathology residents' performance using the metrics of lymph node retrieval and tissue resubmission rates. Lymph node retrieval was calculated by retrospective review of surgical pathology reports from a sample of axillary dissection, mastectomy, and colorectal specimens. Tissue resubmission rates were calculated by retrospective review of a sample of general surgical pathology reports. Pathologists' assistants retrieved a significantly greater total number of lymph nodes compared with pathology residents; however, there was no difference in the total number of positive lymph nodes retrieved. Cases for which pathologists' assistants performed the gross examination had a significantly decreased resubmission rate compared with those performed by residents. In this setting, the gross examination performance of pathologists' assistants was equivalent to or superior to that of pathology residents. These results provide the first information available relating to pathologists' assistants' performance in surgical pathology.

Clinical perception of disease probability associated with Bethesda System diagnoses.

The degree to which clinical perceptions of Papanicolaou smear sensitivity contribute to patient mismanagement is uncertain. A voluntary, anonymous questionnaire was mailed to 350 obstetricians/gynecologists (OGYNs) and 350 other primary care providers (PCPs) located in Pennsylvania or Ohio. The clinicians estimated the probability of no disease, dysplasia, and invasive carcinoma for 1 of 7 Bethesda System diagnoses. Differences in probability estimates between provider types and between the clinicians and medical literature data were measured. The response rate was 22.7%. Compared with published values, clinicians estimated similar disease probabilities for many diagnoses. However, for some diagnoses, the probability estimates differed considerably from published values (e.g., overestimation of dysplasia and invasive carcinoma for benign diagnoses and underestimation of dysplasia for some dysplasia diagnoses), and such errors could contribute to patient mismanagement. OGYNs generally were more accurate in probability estimates than PCPs. Methods to convey more accurately these diagnostic disease probabilities should be examined.

Cost-benefit value of microscopic examination of intervertebral discs.

OBJECT: Given the virtual absence of histologically detected, clinically unsuspected disease in intervertebral disc specimens, some authors have advocated that histological examination be discontinued. However, the examination of intervertebral disc specimens remains common practice in most pathology laboratories. No cost-benefit analysis of this practice has been made; therefore, the authors' goal in this study was perform such an analysis. METHODS: Using the University of Iowa surgical pathology database, 1109 patients who had undergone a laminectomy were identified retrospectively. These cases were classified into four categories based on the patients' preoperative clinical diagnosis and final histopathological diagnosis: insignificant clinical diagnosis/insignificant pathological diagnosis (ICIP), significant clinical diagnosis/insignificant pathological diagnosis (SCIP), significant clinical diagnosis/significant pathological diagnosis (SCSP), and insignificant clinical diagnosis/significant pathological diagnosis (ICSP). A significant clinical diagnosis was defined as one other than a benign, noninfectious indication for laminectomy. A significant pathological diagnosis was a diagnosis other than degenerative changes. The cost-benefit value of performing a histological examination in cases with significant or insignificant clinical diagnoses was examined. The cases were classified as: 1068 ICIP, 17 SCIP, 21 SCSP, and three ICSP. On chart review, in all three cases of ICSP an epidural abscess was identified perioperatively and the subsequent histological diagnosis did not affect patient care. The costs per case of identifying a significant pathological diagnosis with a significant and an insignificant clinical diagnosis were $44.79 and $8811, respectively. CONCLUSIONS: Histological examination of intervertebral disc specimens is cost beneficial only if there is a significant preoperative clinical diagnosis.

Implications of prognostic markers in brain tumors.

Summarizing the current evidence regarding the usefulness of the previous markers for predicting patient outcome, the most promising proliferation marker for predicting patient outcome in patients with brain tumors appears to be KI-67/MIB-1. Its potential usefulness appears to be greatest (1) for grade II and III astrocytic and oligodendroglial tumors in adults, where it may potentially predict length of survival; (2) for nonpilocytic gliomas in children, where it may also potentially predict survival; and (3) for benign, completely resected meningiomas, where it may potentially predict tumor recurrence. Although MIB-1 shows potential, we do not believe there is evidence yet definitely to consider MIB-1 labeling index a predictor of prognosis in these tumors due to the lack of published prospective studies validating the preliminary findings of multiple investigators who have performed only single-center retrospective studies. We also believe that the exact use of MIB-1 labeling index as an independent prognostic indicator for any type and grade of tumor may be complex, given the fact that all of these brain tumors have multiple independent prognostic factors contributing to patient outcome, many of which are readily available clinical factors. In the present managed care environment, MIB-1 labeling should probably await demonstration that it significantly contributes to the physician's ability accurately to predict patient outcome. The presence of telomerase RNA or telomerase activity appears to correlate with degree of malignancy in multiple types of brain tumors, including gliomas; however, it currently has no use as an independent prognostic indicator of patient outcome. It may instead be a marker for malignant tumor initiation or progression. p53 and EGFR are molecular markers that show promise as prognostic indicators of recurrence-free and overall survival in patients with GBMs, but further prospective studies are needed to confirm the retrospective findings. Postsurgical evaluation of these markers is potentially helpful in planning follow-up and treatment for these patients, those patients having tumors expressing relatively high levels of these markers requiring closer follow-up and, when possible, more aggressive therapies. Despite intensive investigation into the expression of molecules regulating apoptosis in brain tumors, no evidence presently exists to support their usefulness as markers of patient outcome. This also applies to measurements of the apoptotic rate itself in human brain tumors. Our overall impression, therefore, is that despite our great desire to find the Holy Grail of patient prognosis in the measurement of a single molecular marker with a precise cut-off value, one has not been identified ... yet! If we allow ourselves to think in terms of organismal biology, it is not surprising that the attempt directly to correlate one or even five (MIB-1, telomerase, BCL-2, p53, EGFR) gene products with a phenomenon as complex as a patient's long-term survival is unrealistic. More likely, multiple pieces of clinical information (which we already know significantly impact patient outcome in patients with brain tumors) will be considered in conjunction with new, scientifically proved molecular information as it becomes available, allowing us to predict, ever more accurately, a given patient's clinical course and outcome.

National practice characteristics and utilization of pathologists' assistants.

OBJECTIVE: To obtain descriptive information regarding the practice characteristics and utilization of US pathologists' assistants. DESIGN: A self-administered, mailed, voluntary, anonymous questionnaire was distributed to a cross-sectional sample of 515 US pathologists' assistants registered as members of the American Association of Pathologists' Assistants. The questionnaire contained items relating to subject demographics, practice characteristics, specific task performance, and amount of time spent per day on the performance of specific tasks. Descriptive statistics were used to describe the data in terms of measures of central tendency and dispersion. RESULTS: The response rate was 66.8%. The majority of questionnaires sent and received were from East Coast regions. Of all respondents, 46.6% were women, 57.6% were less than 40 years old, and 60.0% had been practicing 10 years or less. Over half (54.0%) had a master's degree. Almost the entire sample reported working 30 or more hours per week, with 43.4% reporting working more than 40 hours per week. The majority reported earning annual salaries between $56 000 and $75 000. Although task analysis of responses revealed a wide range of responses, the majority of the sample reported spending most of their daily time performing surgical specimen gross examinations (median 300 min/d). Approximately half of respondents also reported spending up to 90 minutes per day on nonspecific tasks such as logging specimens and answering the phone. Most respondents reported spending more daily time on such nonspecific tasks than on autopsy prosection or research. CONCLUSIONS: To our knowledge, this national survey provides the first description of pathologists' assistants across the United States. These data provide a useful tool for tracking changes in the profession.

Effect of Lean method implementation in the histopathology section of an anatomical pathology laboratory.

BACKGROUND: In the USA, the lack of processes standardisation in histopathology laboratories leads to less than optimal quality, errors, inefficiency and increased costs. The effectiveness of large-scale quality improvement initiatives has been evaluated rarely. AIM: To measure the effect of implementation of a Lean quality improvement process on the efficiency and quality of a histopathology laboratory section. METHODS: A non-concurrent interventional cohort study from 1 January 2003 to 31 December 2006 was performed, and the Lean process was implemented on 1 January 2004. Also compared was the productivity of the Lean histopathology section to a sister histopathology section that did not implement Lean processes. Pre- and post-Lean specimen turnaround time and productivity ratios (work units/full time equivalents) were measured. For 200 Lean interventions, a 5-part Likert scale was used to assess the impact on error, success and complexity. RESULTS: In the Lean laboratory, the mean monthly productivity ratio increased from 3439 to 4074 work units/full time equivalents (p<0.001) as the mean daily histopathology section specimen turnaround time decreased from 9.7 to 9.0 h (p = 0.01). The Lean histopathology section had a higher productivity ratio compared with a sister histopathology section (1598 work units/full time equivalents, p<0.001) that did not implement Lean processes. The mean impact, success and complexity of interventions were 2.4, 2.7 and 2.5, respectively. The mean number of specific error causes affected by individual interventions was 2.6. CONCLUSION: It is concluded that Lean process implementation improved efficiency and quality in the histopathology section.

Activation of astrocytes in the spinal cord of mice chronically infected with a neurotropic coronavirus.

Mice infected with the neurotropic JHM strain of mouse hepatitis virus (MHV-JHM) develop a demyelinating encephalomyelitis several weeks after infection. Astrogliosis and infiltration of inflammatory cells are prominent findings in the brains and spinal cords of infected mice. In this report, astrocytes in infected spinal cords were analyzed for expression of three pleiotropic cytokines, TNF-alpha, IL-1 beta, and IL-6; Type 2 nitric oxide synthase (iNOS); and MHC class I and II antigen. The data show that all three cytokines and iNOS are expressed by astrocytes in chronically infected spinal cords. These activated astrocytes are localized to areas of virus infection and demyelination, although most of the astrocytes expressing these proteins are not MHV-infected. MHC class I and II antigen can be detected in these spinal cords as well, but not in cells with the typical morphology of astrocytes. TNF-alpha, IL-6, and iNOS are also evident in the brains of mice with MHV-induced acute encephalitis, but in marked contrast to the results obtained with the chronically infected mice, most of the cells expressing these cytokines or iNOS had the morphology of macrophages or other mononuclear cells and very few appeared to be astrocytes. Additionally, astrocytes and, most likely, oligodendrocytes are infected in the spinal cords of mice with chronic demyelination. These results are consistent with a role for both viral infection of glial cells and high localized levels of proinflammatory cytokines and nitric oxide in the demyelinating process in mice infected with MHV-JHM. They also show that analogously to the human demyelinating disease, multiple sclerosis, astrocytes are a major cellular source for these cytokines in mice with chronic, but not acute disease.

Expression of monocyte chemoattractant protein (MCP-1) and nitric oxide synthase-2 following cerebral trauma.

Traumatic injury to the brain initiates multiple interrelated processes that involve parenchymal, vascular, and infiltrating inflammatory cells. Nitric oxide (NO) and chemokines have been implicated as regulators of the central nervous system injury response. Following a cryogenic lesion of the cerebral cortex in mice, mRNA for NO synthase (NOS)-2 was detected by reverse transcriptase polymerase chain reaction ipsilaterally 12 h after injury and persisted for 2 weeks. While mRNA was also detected contralaterally, the time course of expression was shorter (1 week). By immunohistochemistry, NOS-2 protein was initially detected ipsilaterally 12 h after injury in infiltrating inflammatory cells. Astroglial cells expressed NOS-2 from 24 to 72 h after injury. The expression of monocyte chemoattractant protein (MCP-1) mRNA peaked at 6 h on the lesion side, remained for 24 h and then declined by 48 h. On the unlesioned side, MCP-1 mRNA was expressed to a much lesser extent and had declined by 24 h. The up-regulation of MCP-1 was relatively specific as a closely related mRNA encoding IP-10 was not significantly increased. These findings implicate a role for NOS-2 and MCP-1 as potential regulators of cellular events following cryogenic cerebral trauma.

Microtubule-associated protein-2: a new sensitive and specific marker for pulmonary carcinoid tumor and small cell carcinoma.

Microtubule-associated proteins (MAPs) are a major component of cytoskeleton family proteins associated with microtubule assembly. MAP-2 has been shown to be specifically expressed in neuronally differentiated cells. Pulmonary neuroendocrine carcinomas such as carcinoid tumors and small cell carcinomas are derived from neuroendocrine cells. We hypothesize that neuroendocrine cells may also express MAP-2, and therefore, MAP-2 may be used as a marker for pulmonary carcinomas of neuroendocrine differentiation. To investigate the utility of using MAP-2 expression to separate pulmonary neuroendocrine from non-neuroendocrine tumors, we examined the expression of MAP-2 immunohistochemically in 100 cases of pulmonary carcinomas. The immunoperoxidase method with antigen retrieval was used to characterize the expression of MAP-2, chromogranin, synaptophysin, and neuron-specific enolase in 25 small cell carcinomas, 25 carcinoid tumors, 25 adenocarcinomas, and 25 squamous cell carcinomas. All tumors were lung primaries. All 25 cases of carcinoid tumors (100%) as well as 23 of 25 cases (92%) of small cell carcinomas were positive for MAP-2. Four of 25 cases (16%) of adenocarcinomas were positive for MAP-2 and synaptophysin. Among the 25 squamous carcinomas, 4 cases (16%) were positive for MAP-2, 2 cases (8%) were positive for synaptophysin, 11 cases (44%) were positive for neuron-specific enolase, and none was positive for chromogranin. In conclusion, MAP-2 is a new sensitive and specific marker for the pulmonary tumors of neuroendocrine differentiation. We recommend that MAP-2 be added to immunohistochemical panels to separate non-neuroendocrine from neuroendocrine lung tumors.

Interobserver variability associated with the MIB-1 labeling index: high levels suggest limited prognostic usefulness for patients with primary brain tumors.

BACKGROUND: The use of the MIB-1 labeling index (LI) as a potential prognostic marker for patients with primary brain tumors is controversial. Many studies advocating its prognostic usefulness have suggested discrete MIB-1 LI cut-off values, above which patients have significantly worse outcomes. However, interobserver variability associated previously with MIB-1 LI calculation has not been reported despite the fact that the degree of interobserver variability impacts the clinical usefulness of such cut-off values. METHODS: MIB-1 LIs were calculated independently using a standardized protocol by six pathologist observers for 50 astrocytic gliomas of varying grades. The level of interobserver agreement was determined by calculating kappa statistics for pairwise pathologist comparisons using MIB-1 LI cut-off values of 2.5%, 5.0%, 8.0%, 11.0%, and 15.0%. Spearman rank correlation coefficients were used to assess the pairwise associations between observer MIB-1 LIs. RESULTS: Although there was general agreement among pathologists regarding whether an MIB-1 LI for a given astroglial tumor was low, moderate, or high based on the analysis of correlation, a high level of interobserver variability was associated with the determination of specific MIB-1 LIs. The highest level of agreement occurred using a cut-off value of 5.0%, with pairwise kappa statistics for this value ranging from 0.52 to 0.80. CONCLUSIONS: The high level of interobserver variability suggests that proposed discrete MIB-1 LI prognostic cut-off values most likely are not useful clinically for predicting outcome for individual patients with primary brain tumors. Further prospective studies are needed investigating the prognostic usefulness of MIB-1 LI ranges that optimize interobserver agreement.

Interleukin 6 promotes vasculogenesis of murine brain microvessel endothelial cells.

Interleukin 6 (IL-6) is a cytokine that acts on a wide range of tissues influencing cell growth and differentiation. Here we show that IL-6 plays a role in the early vascular development (vasculogenesis) in the central nervous system (CNS). We report that IL-6 induces the proliferation of brain microvascular endothelial cells in vitro. Furthermore, IL-6 significantly accelerates the formation of tube-like structures by these cells in Matrigel basement matrix. Moreover, IL-6 mRNA is expressed in vivo in two physiological conditions in which vascularization in the CNS is important: (1) during normal brain development, (2) during the healing process of a traumatic brain injury. Expression of IL-6 mRNA coincides with the expression of vascular endothelial growth factor (VEGF) mRNA in the developing brain with decreasing expression following birth. However, IL-6 mRNA can be detected in the healing adult murine brain tissue by in situ hybridization coinciding with the period of intense tissue reorganization. The transient upregulation of IL-6 mRNA during normal brain development and at brain injury site and the effect of IL-6 on in vitro vasculogenesis suggest that IL-6 may play a role in normal physiology of vascularization in the CNS.