Effect of tissue processing on the ability to recover nucleic acid from specific renal tissue compartments by laser capture microdissection

The anatomic heterogeneity of the nephron poses obstacles to microdissection of individual renal compartments for analysis of gene expression. We have systematically analyzed the effects of fixation time and nuclear staining on the ability to recover nucleic acid from individual renal compartments by laser capture microdissection (LCM). Formalin-fixed kidney sections from Wistar rats and archival human renal biopsies were used for DNA analysis. From 1 to 10 individual glomeruli and from 1 to 10 individual proximal tubules were captured sequentially onto polymer films. DNA for beta-globin could be amplified by PCR from even a single glomerulus or tubule. Optimal conditions for DNA amplification were brief (1- or 2-day) formalin fixation. Use of nuclear counterstains, including Weigert's hematoxylin, Harris's hematoxylin, Mayer's hematoxylin, or methyl green, did not adversely affect the ability to extract and amplify DNA. For RNA extraction, glomeruli and tubules were microdissected from renal cryostat sections stored for up to 6 months. By RT-PCR, mRNA expression of the glomerulus-specific gene, Wilms' tumor-1, was identified in as few as 5 microdissected glomeruli and of the tubule-specific gene, aminopeptidase N, in as few as 5 microdissected tubules, with no cross-contamination between renal compartments. Our findings indicate that the LCM method can successfully microdissect pure glomerular and tubular tissue compartments and that the optimal fixation and staining conditions are those employed routinely for renal biopsies, namely overnight formalin fixation and hematoxylin counterstain for DNA extraction, and cryostat sectioning with hematoxylin counterstain for RNA extraction. The specificity of LCM coupled with the sensitivity of RT-PCR should prove a powerful tool for the analysis of gene expression in specific renal compartments from archival human renal biopsies.     

Potential of Reduction in Total Tumor Volume Measured with 3D‐MRI as a Prognostic Factor for Locally‐Advanced Breast Cancer Patients Treated with Primary Chemotherapy

Abstract: For accurate assessment of the response to primary chemotherapy (PCT) for locally advanced breast cancer, we measured reduction in total tumor volume (TTV) by using three‐dimensional magnetic resonance imaging (3D MRI), and examined the relationship between this reduction and patient prognosis. Fifty‐one patients with locally advanced breast cancer were treated with four cycles of docetaxel (60 mg/m²) before surgery. Tumor size was measured with calipers, ultrasonography (US) and conventional two‐dimensional (2D) MRI before and after chemotherapy. TTV was measured with 3D MRI. These and other clinicopathological parameters were statistically analyzed to determine the prognosis for the patients. Median follow‐up time was 46 months (1–64 months). Of the 51 patients, 25 developed distant recurrences. Patients whose TTV decreased by 75% or more after PCT showed significantly better prognosis than others, while tumor size measured with calipers, US and 2D MRI showed no significant relationship with patient prognosis. Of the clinicopathological parameters, only reduction in TTV and histological grade showed a significant association with distant recurrence‐free survival (p = 0.03 and 0.02, log‐rank test), while stepwise multivariate Cox’s proportional hazards analysis identified TTV as the strongest independent prognostic factor. Reduction in TTV measured with 3D MRI can be a useful prognostic factor for patients with locally advanced breast cancer treated with PCT.     

Epidermal growth factor in the urine of patients with renal cell carcinoma and bladder tumor]

We evaluated the concentrations of immunoreactive epidermal growth factor (EGF) in urine samples from 14 patients with renal cell carcinoma, 16 patients with bladder tumor and 43 non-malignant controls by using radioimmunoassay. In the non-malignant controls, urinary EGF excretion significantly decreased with age (r = -0.46, p less than 0.01), and females excreted significantly more EGF than males [16.3 +/- 7.6 and 9.9 +/- 6.0 (mean +/- SD) ng/mg.creatinine; p less than 0.05]. There was no significant difference between urinary EGF excretion in the patients with renal cell carcinoma and non-malignant controls matched for sex and age (15.9 +/- 12.0 and 14.5 +/- 7.9 ng/mg.creatinine). The difference in excretion of EGF between the patients with bladder tumor and non-malignant controls also was not significant (9.9 +/- 5.6 and 11.7 +/- 7.0 ng/mg.creatinine). These findings indicate, that urine EGF has little usefulness as a tumor marker for renal cell carcinoma and bladder tumor.     

Effect of biological response modifiers on a spontaneous murine renal cell carcinoma regression of metastases caused by the streptococcal preparation OK-432

The effect of the streptococcal preparation OK-432, which is one of the biological response modifiers, was examined in BALB/c mice using a transplantable murine renal cell carcinoma (Renca) of spontaneous origin, and an analysis of effector cells was performed. The tumor grew progressively and metastasized consistently to the abdominal lymph nodes and then to distant organs following the inoculation of Renca cells in the left renal subcapsular site in BALB/c mice, and the survival time of the mice was under 42 days. In this tumor model, i.p. administration of OK-432 after tumor inoculation significantly extended the survival time and significantly inhibited the formation of the inoculated tumor itself. Removal of the left kidney on the 7th day after tumor inoculation neither extended the survival time nor augmented the effect of OK-432. Splenic cells obtained on the 7th day after tumor inoculation from Renca-bearing mice treated with OK-432 were capable of lysing syngeneic Renca cells, NK-sensitive allogenic YAC-1 cells, and LAK-sensitive EL-4 cells in a 4-hour 51Cr-release assay in vitro. Those obtained from healthy mice treated with OK-432 also showed cytotoxic activity against Renca cells. The cytotoxicity of splenic cells from Renca-bearing mice treated with OK-432 was lost almost completely for both Renca and YAC-1 cells after in vitro treatment with anti-asialo GM1 antibody, and was partially lost after in vitro treatment with anti-Thy-1,2 antibody. Additionally, in vivo i.p. administration of anti-asialo GM1 antibody significantly counteracted the effect of OK-432 on survival. These findings demonstrated that Renca cells were NK-sensitive and that the i.p. administration of OK-432 was beneficial for the prevention of the spontaneous metastasis of Renca carcinoma. As the effectors, NK cells played a dominant role and activated T cells were also involved.     

The preparticipation physical examination for sports

Up to 25 million children and adolescents in the United States participate in some type of organized sports activity each year. Most of these young athletes receive an annual preparticipation physical examination. The purpose of the examination is to identify any conditions that might interfere with the athlete's ability to participate in a given sport. The criteria for disqualification are different for each sport.     

Association between time to surgery and 90-day mortality after hip fracture: A retrospective cohort study of 1734 cases

Background

It remains unclear whether early surgical intervention can reduce mortality after surgery in hip fracture patients. The aim of this study was to investigate the association between time from injury to surgery and mortality rate within 90 days after hip fracture surgery.

Novel eosinophilic neuronal cytoplasmic inclusions in the external cuneate nucleus of humans

We report the occurrence of neuronal cytoplasmic inclusions (NCIs) in the external cuneate nucleus of humans. The NCIs appeared as accumulations of eosinophilic rod‐like structures in the neuronal somata in 20 (9.5%) of 211 consecutive autopsy cases. Histochemically, the NCIs were stained bright red with Gomori trichrome, Azan‐Mallory and methyl green‐pyronin, indicating that they contain protein and RNA. Immunohistochemically, the NCIs were positive for stress granule marker proteins, including Hu‐antigen R, eukaryotic translation initiation factor 3 and poly(A)‐binding protein 1, but negative for ubiquitin‐ and autophagy‐related proteins. Ultrastructurally, the NCIs were composed of randomly oriented arrays of parallel fibrillar crystalline material with a well‐defined substructure consisting of longitudinal striations, and were often associated with ribosome‐like granules. These NCIs are morphologically, immunohistochemically and topographically distinct from any other inclusions previously described. Their incidence was found to increase with age. A high incidence was also observed in individuals with noninfectious inflammatory disease. These findings suggest that eosinophilic NCIs in the external cuneate nucleus are novel inclusions and might be formed under stress conditions.     

Molecular characterization of a cephamycin-hydrolyzing and inhibitor-resistant class A beta-lactamase, GES-4, possessing a single G170S substitution in the omega-loop

The nosocomial spread of six genetically related Klebsiella pneumoniae strains producing GES-type beta-lactamases was found in a neonatal intensive care unit, and we previously reported that one of the six strains, strain KG525, produced a new beta-lactamase, GES-3. In the present study, the molecular mechanism of cephamycin resistance observed in strain KG502, one of the six strains described above, was investigated. This strain was found to produce a variant of GES-3, namely, GES-4, which was responsible for resistance to both cephamycins (cefoxitin MIC, >128 microg/ml) and beta-lactamase inhibitors (50% inhibitory concentration of clavulanic acid, 15.2 +/- 1.7 microM). The GES-4 enzyme had a single G170S substitution in the Omega-loop region compared with the GES-3 sequence. This single amino acid substitution was closely involved with the augmented hydrolysis of cephamycins and carbapenems and the decreased affinities of beta-lactamase inhibitors to GES-4. A cloning experiment and sequencing analysis revealed that strain KG502 possesses duplicate bla(GES-4) genes mediated by two distinct class 1 integrons with similar gene cassette configurations. Moreover, the genetic environments of the bla(GES-4) genes found in strain KG502 were almost identical to that of bla(GES-3) in strain KG525. From these findings, these two phenotypically different strains were suggested to belong to a clonal lineage. The bla(GES-4) gene found in strain KG502 might well emerge from a point mutation in the bla(GES-3) gene harbored by its ancestor strains, such as strain KG525, under heavy antibiotic stress in order to acquire extended properties of resistance to cephamycins and carbapenems.