Conclusions from a study of venous invasion in stage IV colorectal adenocarcinoma

AIMS: Venous invasion is an established predictor of prognosis in colorectal cancer (CRC). The reported incidence of venous invasion in CRC specimens varies between 10% and 89.5%, mainly as a result of interobserver variability and differences in specimen processing (for example, staining with haematoxylin and eosin (H+E) alone versus the addition of an elastic fibre stain). This study was performed with three purposes in mind, namely: (1) To assess and compare the incidence of venous invasion diagnosed on H+E stained tissue versus tissue stained with both H+E and an elastic fibre stain. (2) To estimate the inherent false negative rate associated with the diagnosis of venous invasion by histopathological evaluation of resected CRC specimens. (3) To compare the resulting data regarding incidence, quantity, site, and type of venous invasion to the pertinent literature. METHODS: Venous invasion was assessed on sections from 81 CRCs resected from patients with synchronous distant metastases (hepatic and non-hepatic). Only stage IV tumours were studied for the following reasons: (1) it can be assumed that in all patients with distant haematogenous metastases venous invasion had occurred, thus enabling the false negative rate to be calculated; (2) there can be no dispute about the clinical relevance of the various characteristics of venous invasion identified in the tumours of patients with synchronous distant haematogenous metastases; and (3) to eliminate the effect of variance in tumour stage on the incidence of venous invasion. Initially, H+E stained sections were studied for venous invasion. Sections that were negative or questionable with regard to venous invasion were then stained with an elastic fibre stain, and a second search for venous invasion was carried out. Venous invasion was characterised by incidence, quantity, type, and site. The chi(2) test for independence was used to compare the incidence of venous invasion in colonic versus rectal and rectosigmoid primary tumours, and in patients with hepatic versus non-hepatic metastases. RESULTS: Venous invasion was identified in 42 (51.9%) (of the 81 specimens on H+E stained sections. The addition of the elastic fibre stain enabled the diagnosis of venous invasion in 15 (38.5%) of the remaining 39 specimens, increasing the overall incidence to 57 of 81 cases (70.4%). Of the 57 positive specimens, venous invasion was minimal in 27 (47.4%), intermediate in five, (8.8%) and massive in 25 (43.9%). Only intramural veins were involved in 18 (31.6%), only extramural veins in 26 (45.6%), and both intramural and extramural veins in 13 (22.8%) of the 57 positive specimens. The filling type of venous invasion was found in 41 (71.9%), the floating type in 28 (49.1%), and the infiltrating type in six (10.5%) of the 57 positive specimens. There was no significant difference between the incidence of venous invasion in the colon (42 of 60; 70%) versus rectal and rectosigmoid tumours (15 of 21; 71.4%; p = 0.8539), nor in the incidence of venous invasion in patients with hepatic (49 of 70; 70%) versus non-hepatic (eight of 11; 72.7%) metastases (p = 0.9018). CONCLUSIONS: The addition of an elastic fibre stain enables the identification of venous invasion in a considerable proportion of sections from CRC tumours that are falsely negative for venous invasion on H+E stain alone. The inherent chance of missing venous invasion on histopathological evaluation of CRC tumours stained with H+E and elastic fibre stains is at least 10.5%, and may be as high as 29.6%. In a large proportion of stage IV CRCs, despite the presence of synchronous distant metastases, only a minimal extent of venous invasion (that is, one to two involved veins) is demonstrable in the primary tumour. This suggests that only minimal venous invasion is required for the seeding of clinically relevant haematogenous metastases, and emphasises the careful, dedicated search for venous invasion that is required from the pathologist. Although extramural venous invasion was predominant in stage IV CRCs, in a considerable proportion of tumours (about a third) only intramural venous invasion was found. This suggests that intramural venous invasion may also seed clinically relevant haematogenous metastases, and should therefore also be considered as an indicator of poor prognosis.     

Antimicrobial activities against 84 Listeria monocytogenes isolates from patients with systemic listeriosis at a comprehensive cancer center (1955-1997)

Listeriosis is a serious complication in patients undergoing treatment for cancer. We present antimicrobial susceptibility profiles of 84 clinical Listeria monocytogenes isolates. During 1955 to 1997, in vitro susceptibility for penicillin (97.6%), ampicillin (90.7%), erythromycin (98.8%), tetracycline (96.9%), and gentamicin (98.0%) remained unchanged. All isolates were susceptible to amikacin, ciprofloxacin, imipenem, rifampin, trimethoprim-sulfamethoxazole (TMP-SMX), and vancomycin. High prevalence of clindamycin resistance (96.2%) was unexpected. Ampicillin plus gentamicin is standard therapy for systemic listerosis, and TMP-SMX may be used for patients with beta-lactam intolerance. In vitro susceptibility profiles for carbapenem and fluoronated quinolone are promising, although clinical validation is critically needed before routine use is advocated, especially for listeric patients with severe cellular immune defects.     

Immunodiagnosis of groundnut and watermelon bud necrosis viruses using polyclonal antiserum to recombinant nucleocapsid protein of Groundnut bud necrosis virus

In vitro gene expression strategy was used for the production of polyclonal antiserum to the nucleocapsid protein (NP) of Groundnut bud necrosis virus (GBNV). The GBNV NP gene from cowpea isolate was cloned into 6x His-tagged UA cloning vector and expressed in Escherichia coli [M15] cells. The fusion protein was detected in insoluble fraction and was purified by using Ni-NTA agarose resin. The purified 6x His-fusion protein (32 kDa) was used for immunisation to produce a high titre polyclonal antiserum. The antiserum to the NP of GBNV at 1:4000 dilution detected successfully natural infection of GBNV and Watermelon bud necrosis virus in a wide range of cucurbitaceous, leguminous and solanaceous hosts from different locations.     

A Cross-cultural Investigation of the Factors and Biases Involved in the Allocation of Scarce Medical Resources

In this study Spanish and English lay participants were given minimal demographic information regarding 16 hypothetical patients, and were required to rank them in order of priority for a kidney dialysis machine. The patients differed in terms of their gender, smoking behaviour, political affiliation and place of birth. These factors were combined factorially, so that each of the 16 patients presented a unique combination of attributes. The English sample showed a preference for local, female, non-smoking patients. The results for the Spanish sample were similar with respect to place of birth and gender, but smoking behaviour was not significant. Neither sample demonstrated a preference on the basis of political affiliation. The results suggest that certain demographic factors may bias the decision making of individuals or committees involved in the allocation of scarce medical resources.     

Extraction and genotyping of Cryptosporidium parvum DNA from fecal smears on glass slides stained conventionally for direct microscope examination

A method was developed for extracting cryptosporidial DNA from stained fecal smears on glass microscope slides. The correct genotype of Cryptosporidium parvum was amplified by PCR from 89 (85%) of 105 smears following conventional staining but not from negative controls. This technique may have applications for analysis of other infectious agents.     

Calcium is required for the expression of anthrax lethal toxin activity in the macrophagelike cell line J774A.1.

Anthrax lethal toxin, which consists of two separate proteins, protective antigen (Mr, 82,700) and lethal factor (Mr, approximately 83,000), is cytotoxic to the macrophagelike cell line J774A.1. Removal of calcium from the culture medium protected cells against the action of lethal toxin. Calcium depletion during the binding phase of intoxication afforded only partial protection. Further analysis showed that calcium removal caused some inhibition of protective antigen binding but that it had minimal effect on proteolytic conversion of protective antigen to the active 63-kilodalton fragment and that it had no effect on lethal factor binding. Cells to which lethal toxin had bound in the presence of calcium were protected when transferred to calcium-depleted culture medium, indicating a role for calcium at a postbinding stage. When ammonium chloride is present with lethal toxin, toxin accumulates in intracellular vesicles. Calcium-free medium protected these cells upon removal of the amine block, suggesting that calcium is also required at a step after internalization of lethal toxin. Calcium channel blockers inhibited 45Ca2+ uptake and protected cells against cytotoxicity. Calmodulin inhibitors also protected against the action of lethal toxin, suggesting involvement of calmodulin at a step during intoxication. We conclude that calcium is required at several steps in the intoxication of cells by anthrax lethal toxin.     

Predicting intellectual outcome among children treated with 35-40 Gy craniospinal irradiation for medulloblastoma

Fifty children diagnosed with medulloblastoma completed 188 psychological evaluations using the Wechsler Intelligence Scales for Children (D. Wechsler, 1974, 1991) over a 7-year study period following 35-40 Gy postoperative craniospinal irradiation. Random coefficient models were used to predict the trend in the children's intellectual performance as a function of time since diagnosis, with both patient and treatment variables as parameters of this function. A quadratic model demonstrated a delay prior to decline in performance for older patients, whereas the younger patients showed an immediate loss of performance with a plateau at approximately 6 years postdiagnosis. A steeper decline was found for those with higher baseline performance. Clinicians may use the proposed predictive model to identify those patients who are at risk of significant intellectual decline.     

Heat stress alters the virulence of a rifampin-resistant mutant of Francisella tularensis LVS.

We have studied the stress response of a rifampin-resistant mutant of Francisella tularensis LVS. This mutant, Rif 7, was avirulent with an intraperitoneally administered 50% lethal dose greater than 10(7) CFU in a murine model of infection. Exposure of Rif 7 to heat stress for 5 h in vitro resulted in a 2-log decrease in its 50% lethal dose (P < 0.02). The increase in virulence was dependent on the time of exposure to high temperature and was maximal at 5 h. Envelope preparations from heat-stressed cells showed increased levels of several proteins. Notable among these were polypeptides with approximate molecular masses of 16, 60, and 75 kDa. Increases in both virulence and envelope protein levels were reversed when heat-treated cells were subsequently grown at 37 degrees C. Inhibition of protein synthesis by actinomycin D during heat stress blocked the increase in virulence of Rif 7. Cell-free media from the heat-stressed Rif 7 reacted with the whole spectrum of bacterial proteins were not toxic to mice. Hyperimmune serum against Rif 7 reacted with the whole spectrum of bacterial proteins in Western blots (immunoblots), although its reaction with 34- and 45-kDa proteins and two 60- and 75-kDa proteins upregulated during heat stress was weak. Other stress conditions, low iron and low pH, caused similar increases in the virulence of Rif 7. However, examination of the protein profile did not reveal any major common polypeptides induced by different stresses. Heat-treated Rif 7 bacteria were fully able to replicate in macrophages in vitro and in the host tissues, even though heat treatment only partially restored virulence.     

Identification of genes differentially expressed in cultured human osteoblasts versus human fibroblasts by DNA microarray analysis

Little is known about patterns of gene expression from cells populating the connective tissues. This study investigated the possible variance of gene expression profile between human osteoblasts (HO) and human fibroblasts (HF) in vitro, using DNA microarray technology. Clustering identification was used to compare expression patterns between HO and HF for biological significance. Our results showed that genes encoding the extracellular matrix or apoptosis-related proteins tended to be expressed in greater abundance in HO, while more proteolysis-related proteins were expressed in higher level in HF. Significant differences in expression were also noted with genes related to signaling pathways. To confirm the array results, three genes (periostin, MFG-E8, MMP-10) were selected and analyzed independently by RT-PCR and northern blot. The results were found consistent with the array data in HO and HF. The present findings suggest that HO and HF differ not only phenotypically but in the expression level of tissue specific genes to assure the turnover and homeostasis of their respective tissues.