Immunohistochemical characterization of an anti-epithelial monoclonal antibody (mAB lu-5)

Summary A mouse monoclonal antibody (mAB lu-5) was prepared using a lung cancer cell line as an antigen. The selected clone produces an IgG with a gamma-1 heavy chain and a kappa-light-chain. Immunohistochemical testing of mAB lu-5 on 117 normal tissue biopsies and 474 tumours revealed reactivity with an intracytoplasmic, formaldehyderesistant antigen present in most epithelial and mesothelial cells, but absent in mesenchymal cells. The antibody can therefore be used as a first order, pan-epithelial marker. It proved also useful for fast tumour diagnosis on frozen sections.     

In vitro effect of ascorbic acid on bovine kidney alkaline phosphatase activity

A striking decrease of bovine kidney alkaline phosphatase activity is observed in vitro when the catalytic assay is performed after preincubation of the enzyme with ascorbic acid (AA). The inhibitory effect is a function of AA concentration time and on temperature. Activity decay follows an exponential biphasic course as a function of preincubation time composed by a "fast" phase in the first half hour and by a later "slow" phase of inhibition. Both the rise in preincubation temperature and the increase of the amount of vitamin enhance the degree of inhibition. Ascorbic acid is ineffective as inhibitor when added together with the substrate, p-nitrophenyl phosphate, which in fact markedly stabilizes the enzyme even when present in unsaturating amounts.     

Low doses of rIL2 after autologous bone marrow transplantation induce a “prolonged” immunostimulation of NK compartment in high-grade non-Hodgkin's lymphomas

Ten patients with high-grade non-HodgKin's lymphoma (HG-NHL) entered a subcutaneous (s.c.) recombinant interleukin 2 (rIL2) trial within 2 months of undergoing autologous bone marrow transplantation (ABMT). Immunological studies, consisting in T- and natural killer (NK)-cell subset assessment, together with functional assays, such as NK activity and CD16-mediated redirected killing assay, were performed before therapy, after 2 weeks, and then monthly. Phenotypic analysis showed a significant increase (p=0.01) of CD16 and CD56 NK cells, from 12% to 28% and from 17% to 37%, respectively. In particular, the CD56^bright NK cell population showed a tenfold increase, while CD56^dim NK cells remained unmodified compared with pretreatment values. The expression of IL2 receptors was also studied, and a significant increase (p=0.01) of CD122 (p75)-positive cells from 8% to 30% was found, while no significant increase was observed in CD25 (p55)-positive cells. Furthermore, rIL2 administration led to an increase of NK activity even at the lowest effectors: target ratio and to an increase of CD16-mediated redirected killing assay. These phenotypic and functional modifications lasted throughout the duration of rIL2 therapy and remained after completion of therapy. In addition, none of the ten patients relapsed, and two of them who started IL2 treatment while still showing residual disease experienced a complete disappearance of the disease after 10 and 7 months of therapy, respectively. Our data suggest that infusion of rIL2 s.c. after ABMT is safe, can selectively increase NK cell number and function, and may have a beneficial effect on the minimal residual disease.This work sas supported in part by CNR PF ACRO: 94.01214.PF39.     

Use of unconventional lipid substrates in parental nutrition

In addition to the classic soybean oil fat emulsion, developed more than 40 years ago and still widely used, emulsions with other lipid substrates are available today for parenteral nutrition; these substrates implement the benefits offered by soybean oil when mixed with it in given proportions. Soybean oil triglycerides are rich in linoleic acid, a long chain omega-6 polyunsaturated fatty acid, which is essential and is an indispensable component of parenteral nutrition. However, very high doses of omega-6 polyunsaturated fatty acids should be avoided, particularly in some critical illnesses. Medium chain triglycerides, long well known to nutritionists and dietitians for their easy intestinal absorption, have become available in parenteral nutrition emulsions in a mixture with soybean oil. Medium chain triglycerides are completely and readily used for energy production and do not interfere significantly in the production of inflammatory mediators, in the composition of cell membranes and in body organ and system functions. Omega-3 polyunsaturated fatty acids, essential fatty acids derived from fish oil, permeate cell structure and affect cell activity with different mechanisms, playing also an important role in the modulation of inflammatory processes. Omega-3 emulsions in parenteral nutrition are currently added as a supplement to other fat emulsions. Knowledge of these "non-conventional" fat emulsions is being continuously improved by investigative work and clinical experience.     

In vivo bone metastases, osteoclastogenic ability, and phenotypic characterization of human breast cancer cells

Mouse bone marrow cells cultured with human breast cancer MCF-7 cell-conditioned media showed osteoclastogenesis with an increment of bone resorption, although conditioned media from an adriamycin-selected MCF-7 clone (MCF-7ADR) had no effect. Consistently, MCF-7 cells induced 5-fold more in vivo experimental osteolytic bone metastases, with no soft tissue lesions, compared to MCF-7ADR cells. Paracrine factors stimulating (interleukin (IL)-6, IL-1beta, tumor necrosis factor-alpha (TNF-alpha)) or inhibiting (IL-12, IL-18, granulocyte macrophage-colony stimulating factor (GM-CSF)) osteoclastogenesis were significantly increased in MCF-7ADR relative to MCF-7 cells, suggesting that the inhibitory cytokines could selectively overwhelm the effects of the stimulatory ones. Treatment of osteoblast primary cultures with MCF-7-conditioned medium induced a selective upregulation of IL-6 expression, suggesting an indirect stimulation of osteoclastogenesis via the osteoblasts. MCF-7 and MCF-7ADR showed no difference in proliferation rate. However, a higher ability to migrate and invade gelatin and matrigel was observed in MCF-7ADR. Enhanced invasiveness might result from increased metalloproteinase (MMP) activity and cytoskeleton rearrangement. MCF-7ADR cells expressed higher levels of c-Src, focal adhesion kinase (FAK), and protein tyrosine kinase 2 (PYK2) involved in cell adhesion and motility. MCF-7 and MCF-7ADR expressed high and faint levels of functional estrogen receptor alpha (ERalpha), respectively. MCF-7ADR also showed significantly higher levels of the protein kinase C (PKC) alpha and beta2 and a selective activation of PKC compared to MCF-7, where the most abundant isoforms were beta1 and delta. Heat shock protein 27 (Hsp27) was more abundant in MCF-7 cells, but failed to translocate to the nucleus in response to heat shock. In conclusion, we have demonstrated that despite the fact that MCF-7ADR cells showed a more invasive phenotype relative to MCF-7, they have low potential to induce osteolytic bone lesions and stimulate osteoclastogenesis and osteoclast activity. Therefore, we believe that reduced aggressiveness of breast carcinomas could correlate with a greater osteolytic activity featuring their bone metastases.     

A new POLG1 mutation with peo and severe axonal and demyelinating sensory–motor neuropathy

Background Progressive external ophthalmoplegia (PEO) is a mitochondrial disorder associated with defective enzymatic activities of oxidative phosphorylation (OXPHOS), depletion of mitochondrial DNA (mtDNA) and/or accumulation of mtDNA mutations and deletions. Recent positional cloning studies have linked the disease to four different chromosomal loci. Mutations in POLG1 are a frequent cause of this disorder. Methods We describe two first–cousins: the propositus presented with PEO,mitochondrial myopathy and neuropathy, whereas his cousin showed a Charcot– Marie–Tooth phenotype. Neurophysiological studies, peroneal muscle and sural nerve biopsies, and molecular studies of mtDNA maintenance genes (ANT1, Twinkle, POLG1, TP) and non dominant CMT–related genes (GDAP1, LMNA, GJB1) were performed. Results A severe axonal degeneration was found in both patients whereas hypomyelination was observed only in the patient with PEO whose muscle biopsy specimen also showed defective OXPHOS and multiple mtDNA deletions. While no pathogenetic mutations in GDAP1, LMNA, and GJB1 were found, we identified a novel homozygous POLG1 mutation (G763R) in the PEO patient. The mutation was heterozygous in his healthy relatives and in his affected cousin. Conclusions A homozygous POLG1 mutation might explain PEO with mitochondrial abnormalities in skeletal muscle in our propositus, and it might have aggravated his axonal and hypomyelinating sensory–motor neuropathy. Most likely, his cousin had an axonal polyneuropathy with CMT phenotype of still unknown etiology.