Properties of wr2721 (ethiofos) as modulator of Cisplatin-induced neurotoxicity studied at the ultrastructural level in the pond snail lymnaea-stagnalis

Modulating effects of WR2721 were studied on cisplatin-induced histological and ultrastructural changes in the ganglia of the central nervous system of the pond snail Lymnaea stagnalis. The relevance of the study was indicated by showing histochemically that alkaline phosphatase, the enzyme converting WR2721 into the active drug WR1065, is present in the snail brain. Central nervous systems of the snail were either preincubated in 5 mM WR2721 or in snail Ringer for 1 h and then postincubated for 10 or 20 h in: (i) Ringer (control), (ii) WR2721 (5 mM), (iii) cisplatin (0.4 mM), or (iv) cisplatin (0.4 mM) plus WR2721 (5 mM). The following parameters were studied: (i) general morphology, (ii) chromatin (number and mean clump size per unit surface ama, clump size frequency distribution), (iii) nucleoli (ratio of granular/fibrillar areas, (iv), Golgi zones (mean surface area, area containing electron dense material), (v) secretory granules (number), (vi) lysosomes (number per unit surface area of cytoplasm). The focus was on two types of identified neuroendocrine cells. Incubation in Ringer alone (controls) caused slight inactivation of the cells between 10 and 20 h of incubation (e.g. relative decrease of the granular part of the nucleoli). WR2721 alone had comparable effects on the tissue. In addition, in this group, a reduction in the electron dense material of the Golgi zones was observed. No major differences in number of secretory granules were observed in any of the groups. Treatment with cisplatin alone for 20 h caused a disappearance of the orange colour of the ganglia, swelling of axons and distension of intercellular spaces. Such changes were not observed in the group treated with cisplatin plus WR2721 or any of the other experimental groups. Both cisplatin alone and cisplatin plus WR2721 caused an increase in the number of chromatin clumps and a decrease in the mean chromatin clump size after 10 and 20 h of incubation. With regard to these parameters no differences were observed between the two groups. The chromatin clump size distribution curves of both groups were significantly different from the curve of the controls. Compared to that of the cisplatin group, the curve of the cisplatin plus WR2721 group, especially after 10 h, had shifted in the direction of that of the controls. Treatment with cisplatin alone induced drastic changes in nucleolar morphology. After 20 h of incubation the nucleoli had transformed into homogeneous dense structures. After 10 h of incubation in cisplatin plus WR2721 nucleoli still had a normal appearance. After 20 h those of the co-treated group had also become electron dense and appeared to contain numerous dark dots. Cisplatin caused a significant decrease in the extent of the Golgi zones. Co-treatment with WR2721 prevented this decrease to a certain degree. In both groups the electron dense material had disappeared from the Golgi zones. Furthermore, cisplatin alone induced an increase in the number of lysosomes. This increase was slightly (but not significantly) prevented by co-treatment with WR2721. The observations on the snail neurons show that WR2721 at the concentration studied induced only minor morphological changes. The drug modulates to some extent the pathological changes caused by cisplatin. The results support clinical data indicating that cisplatin-induced neurotoxicity is reduced by WR2721.     

A novel small-cell lung carcinoma-specific DNA-binding activity related to the transcription factor ap-1

A novel TRE-binding protein complex was detected specifically in 12 out of 13 small cell lung carcinoma (SCLC) cell lines. This complex was characterised by a lower electrophoretic mobility than the 'ubiquitous' complex present in all other carcinoma cell lines analysed. As shown by UV-crosslinking and South-Western blotting, the SCLC-specific complex contains a protein with an apparent M(r) > 100 kD, which is far bigger than all Fos and Jun proteins described to date. In addition, the DNA-binding specificity of this complex is different from the specificity of the 'ubiquitous' complex or a Fos/Jun heterodimer.     

The effect of infused fat emulsions on reticuloendothelial function in the rabbit

The effect of the infusion of different fat emulsions (Intralipid and MCT/LCT mixtures) on the reticuloendothelial function of the rabbit has been investigated. Emulsions containing 20% dispersed triglyceride were administered over 6 h to a total of 3 g/kg body weight. The extent of blockade of the reticuloendothelial system was measured using a labelled probe in the form of technetium-99m labelled albumin microspheres. Scintigraphic and blood and organ level determinations demonstrated that all emulsions caused an impairment of reticuloendoethlial function, but this was small.     

Differential expression of the neu transgene in murine mammary tissues

The mammary glands of control FVB and mice with MTV-LTR promoted transgenes were stained using immunohistochemistry to detect neu expression. Neu expression in the terminal end buds of developing mammary glands and during early pregnancy in FVB mice was confirmed by in situ hybridization. Neu was expressed in all tumors from mice with the neu transgene but not in tumors expressing transforming growth factor alpha (TGF alpha) or polyoma virus middle T (PyV-MT). Neu was also expressed sporadically in non-neoplastic mammary cells of transgenic neu mice. However, most mammary cells expressing neu were dysplastic. The differential expression of the neu transgene has important implications for the interpretation of transgenic biology.     

Correlation between apoptosis and tumor-associated macrophages in human invasive ductal breast carcinoma

The relationship between apoptosis and tumor-associated macrophages (TAM) was studied in situ in 60 breast carcinomas (18 G1, 28 G2 and 14 G3 carcinomas) using simultaneous apoptosis (TUNEL method) and macrophage staining (anti-CD68 antibody). Apoptotic tumor cell rate (ATCR), total TAM content (TAM(TOT)) and the proportion of TAM that had either cell-to-cell contact with apoptotic tumor cells or that were phagocytosing them (TAM/APO cells) were quantified. ATCR correlated significantly with TAM(TOT). Within all apoptotic tumor cells, the proportion of TAM/APO cells was lower than 20%. Considering the fact that cell-to-cell contact is essential for macrophage-mediated tumor cell killing, our data suggest that the majority of apoptoses occurring in breast cancer may not be caused by macrophage tumoricidal activity. TAM/APO cells accounted for only 1.7% of all TAM. Thus, tumor cell killing and apoptotic tumor cell phagocytosis seem to be quantitatively less important functions of TAM in human breast cancer in vivo.     

Aspergillosis of the cranial base

Aspergillus is an ubiquitous organism seldom pathogenic in normal hosts. Aspergillus osteomyelitis of the spine occurs rarely in immunocompromised patients as a result of hematogenous spread from distant foci. We present a case of Aspergillus osteomyelitis in the region of the jugular foramen in a previously healthy male with no antecedent event. He presented with dysphagia, hypophonia, and weight loss of several months duration. Diagnosis was delayed due to nonspecific results of various imaging tests. We review the clinical course of fungal osteomyelitis, including appearance on magnetic resonance imaging and computed tomography, culture characteristics, and gross appearance. Current treatment consists of surgical debridement and antifungal medications such as amphotericin B and itraconazole, and the efficacy of these are discussed.     

Rflp analysis of the L-myc oncogene in head and neck-cancer - relationship study with susceptibility and disease progression

The L-myc DNA restriction fragment length polymorphism (RFLF), revealed by EcoRI digestion, has been evaluated in a case-control study including 161 head and neck cancer (HNSCC) patients and 160 normal healthy individuals with similar smoking and alcohol habits. No significant difference in the distribution of L-myc genotypes (LL, LS or SS) was found between the two populations implying thus no predisposition to head and neck tumour by either allele. There was no significant association between L-myc genotypes and the usual clinicopathological features such as T staging, differentiation status and lymph node involvement. Moreover, follow-up data from 154 patients was obtained and correlated with the L-myc pattern. No significant difference was observed in metastasis occurrence, multiple cancer incidence and survival data in the patients classified according to the L-myc genotypes; only a trend to preferentially develop metastasis in lung for patients with S allele was noted. In conclusion, our data shows that the L-myc typing does not contribute to HNSCC risk or prognosis assessment. A review of L-myc RFLP published studies shows contradictory results even on the same type of tumour and emphasizes the lacunae in understanding the biological role of L-myc for valid interpretation of L-myc allelic associations with cancer susceptibility or prognosis.     

Alteration of the C-met oncogene locus in human head and neck-carcinoma

The c-met proto-oncogene encodes the receptor for the hepatocyte growth factor/scatter factor (HGF/SF) which induces eel proliferation and motility. We have analysed the genetic alteration involving the c-met locus on chromosome 7q31 using the pMet H polymorphic probe, in tumor and normal DNA from 87 patients with head and neck squamous cell carcinomas (HNSCC). We report the observation of loss of heterozygosity (LOH) at this locus in 23% of informative cases, contrasting with the previously reported 40% LOH detected in breast cancer. Further, gain of genetic material was also observed in 13% of the HNSCCs. The alterations of c-met gene were not significantly associated with standard pronostic features including tumor size and lymph node status. Involvement of the c-met locus in allelic imbalance, either loss or gain of genetic material, is relatively consistent with complex karyotype patterns detected in head and neck squamous cell carcinomas through previous cytogenetic studies.     

Migration of leukocytes across endothelium and beyond: molecules involved in the transmigration and fate of monocytes

Passage of leukocytes across the endothelial lining into sites of inflammation has been shown to be regulated largely by platelet/endothelial cell adhesion molecule-1 (PECAM/CD31). We summarize recent work from our laboratory documenting that PECAM is involved both in diapedesis and the subsequent step of migration across the basal lamina. The former process involves a homophilic interaction between the amino-terminal extracellular domains of PECAM on the leukocyte and on the endothelial cell. The latter process involves a heterophilic interaction between the membrane-proximal extracellular domain of PECAM and an unknown ligand(s) in the subendothelial basal lamina. These findings are demonstrated in both in vitro and in vivo models. For monocytes, however, transmigration is just the first step in the next phase of their lives. In addition to their specific recruitment during the inflammatory response, many monocytes constitutively leave the bloodstream to enter tissues. However, only a fraction of these become tissue macrophages. In an in vitro model of monocyte emigration, approximately half of the leukocytes that initially transmigrate an endothelial monolayer migrate back out in the basal-to-apical direction within the next 2 days. This reverse transmigration cannot be blocked by anti-PECAM reagents, but involves p-glycoprotein and tissue factor expressed on the leukocytes. The reverse transmigrating cells are phenotypically dendritic cells (DC). Their maturation to mature DC can be accelerated by inclusion of inflammatory stimuli in the co-culture. The cells that remain behind in the subendothelial collagen are phenotypically macrophages. We postulate that a major source of DC in lymph nodes is cells derived from monocytes that enter a tissue via the blood and leave several days later via afferent lymphatic channels.     

Simple, Rapid Enzyme-Linked Immunosorbent Assay (ELISA) for the Determination of Rat Osteocalcin

Determination of the concentration of osteocalcin in rat serum is frequently performed using a commercially available radioimmunoassay (RIA). However, this assay takes 3 days to complete, uses radioactive material, and has a narrow linear range. The limited range of the RIA makes it necessary to test multiple dilutions of the sample which frequently results in values that differ, depending on the dilution. In order to overcome these limitations, we have developed an ELISA that utilizes the same standards and anti-rat osteocalcin antiserum, as is used in the RIA. The principle of the ELISA is that the osteocalcin in the sample competes with osteocalcin previously immobilized on a microtiter plate to bind to the available anti-rat osteocalcin antibodies. The amount of antibody bound to the immobilized osteocalcin is determined colorimetrically using a secondary antibody coupled to alkaline phosphatase. This ELISA has a three-log linear response with a sensitivity of 0.1–0.15 ng/ml and intra- and interassay coefficient of variance (CV) values of less than 10%. Most importantly, the assay is rapid and only requires a 2-hour incubation of the sample with the antiserum. The incubation time is important since we and others have observed a significant decrease in the osteocalcin level from serum samples incubated for long periods of time with the antiserum, presumably due to degradation of the osteocalcin. In general, the commercially available RIA gives osteocalcin values that are one-half to one-fourth that of the ELISA because the RIA requires a 48-hour incubation time. Received: 14 November 1997 / Accepted: 9 July 1998