T-cell activation marker expression on tumor-infiltrating lymphocytes as prognostic factor in cutaneous malignant melanoma.

The central role of T cells in antitumor immunity is well established. However, tumor progression, often seen in the presence of substantial lymphocytic infiltration, suggests that these T cells are not capable of mounting an effective immune response to control tumor growth. Evidence has accumulated that T lymphocytes infiltrating human neoplasms are functionally defective, incompletely activated, or anergic. Therefore, when characterizing the immune competent cells within lymphoid infiltrates of tumors, it is important to assess their activation state. We investigated the expression of two T-cell activation markers, interleukin 2 receptor alpha (CD25) and OX40 (CD134), by immunohistochemistry in primary cutaneous melanoma samples of 76 patients and analyzed it in relation to tumor stage and tumor progression (>5 years follow-up), as well as to patients' survival. We found that the degree of infiltration by CD25(+) and intratumoral OX40(+) lymphocytes showed a tendency to decrease in thicker melanomas. The frequency of samples with high numbers of peritumoral CD25(+) and OX40(+) cells was significantly lower (P = 0.0009 and P = 0.0087, respectively) in melanomas developing distant visceral metastases, compared with nonmetastatic or lymph node metastatic tumors. For both activation markers studied, high peritumoral densities were associated with longer survival by univariate analysis (P = 0.0028 and P = 0.0255 for CD25 and OX40, respectively), whereas peritumoral OX40(+) lymphocyte infiltration had an impact on survival also in multivariate analysis (P = 0.035). The results suggest that the presence of lymphocytes expressing the T-cell activation markers CD25 or OX40 shows correlation with tumor progression as well as with patients' survival in cutaneous malignant melanoma.     

Cutaneous Blood Flow and Peripheral Resistance in Type II Diabetes as Compared to Intermittent Claudication Patients

Insulin deficient, type I diabetic patients have reduced skin blood flow reserve. It is not known whether these skin perfusion abnormalities also exist in non-insulin dependent (type II) diabetic patients. An additional open question is whether the reduced skin blood flow is due to increased resistance of the cutaneous microvasculature or to decreased peripheral perfusion pressure due to increased atherosclerosis in the diabetic population. We measured skin blood flow by laser Doppler flowmetry in patients with type II non-insulin treated diabetes. Limb systolic blood pressure was measured distally using a sensitive sonar Doppler device at the finger and toe. The ratio of pressure to flow was computed as an index of peripheral blood flow resistance. To assess the effect of cutaneous blood flow resistance, we elicited maximal vasodilation by increasing local skin temperature directly at the site of the laser Doppler probe. We compared blood flow and pressure in diabetic patients with the values in non-diabetic control patients. As a further control population, we also assessed these same parameters in non-diabetic patients with peripheral vascular disease, which may be expected to decrease large arterial blood flow pressure without directly affecting the microvasculature. There were 68 type II diabetic patients, 18 non-diabetic control subjects, and 25 non-diabetic patients with intermittent claudication. We measured skin blood flow at the dorsal surfaces of the finger and toe, sites with primarily nutritive capillary perfusion, and at the plantar surfaces of the finger and toe, where arteriovenous shunt perfusion predominates. Heat stimulated flow was markedly lower for the diabetic patients at the finger dorsal surface (16.5 ± 1.4 ml/min/100 g vs 29.8 ± 4.4 ml/min/100 g in the non-diabetic group (p < 0.05). The resistance index was 13.2 ± 1.9 in the diabetic patients and 6.8 ± 1.7 in the controls (p < 0.05). At the toe dorsum, basal temperature flow was significantly lower in the diabetic group (0.6 ± 0.1 ml/min/100 g) than in the non diabetic group (1.1 ± 0.2 ml/min/100 gm) with resistance index almost twice as high (379 ± 32) in the diabetic group versus non-diabetic controls (208 ± 36) [p < 0.01 for both comparisons]. With the local application of heat, there was a much larger increase in flow in the non-diabetic subjects than in the diabetic group. The resistance index dropped much more with heat stimulation for the non-diabetic patients (10.8 ± 3.3) than for the diabetic patients (50.6 ± 10.4) [p < 0.01] There was a lesser rise in flow at the toe pulp surface with heat in the diabetic patients (31.3 ± 3.0 ml/min/199 gm) than in the control subjects (45.4 ± 5.9 ml/min/100 gm; p < 0.05) with a higher resistance index (13 ± 4) than in the non-diabetic subjects (4 ± 1) [p < 0.05]. The claudication patients had substantially greater flow at the toe dorsal surface at basal temperature (2.2 ± 0.4 ml/min/100 gm) with significantly lower resistance index (126 ± 24) than the non-diabetic controls (p < 0.05). At 44°C, toe dorsum flow was significantly higher (17.8 ± 3.7 ml/min/100 gm) than in the diabetic patients with lower resistance index (17.0 ± 6.6) [p < 0.05]. Toe pulp flow at basal temperature was significantly higher (10.1 ± 2.0 ml/min/100 gm) than in either the diabetic (3.8 ± 0.6) or non-diabetic control groups (3.5 ± 1.4) [p < 0.05]. Skin blood flow is impaired in diabetes. The reduction is due to increased resistance in the capillary bed rather than to reduced perfusion pressure. The increased resistance was found only in the diabetic patients, not in the non-diabetic patients with peripheral vascular disease. To the contrary, there appeared to be a compensatory decrease in skin flow resistance in the patients with peripheral vascular disease. Thus, there is a small vessel disease which impairs cutaneous perfusion in diabetes, but there is no such effect on skin blood flow in non-diabetic patients with large vessel disease.     

Expression of CD44v3 protein in human endothelial cells in vitro and in tumoral microvessels in vivo

The most universal angiogenic cytokines (VEGF, bFGF, HGF) are all heparin-binding proteins, the function of which is dependent on cell surface heparan sulfate proteoglycans (HSPG). Several proteoglycans have been demonstrated in endothelial cells, but only glypican-1 from the cell surface HSPG subfamily was documented at protein level. Here, we show that CD44v3 is expressed in human immortalized endothelial cells [anchorage-dependent human umbilical vein endothelial cells (HUVEC) and anchorage-independent Kaposi sarcoma (KS-Imm)] at mRNA and protein level, but is absent from the primary culture of human brain microvascular endothelial cells. We have shown that CD44v3 has a large cytoplasmic pool in endothelial cells, but a limited surface expression, mainly at filopodia, colocalized with MMP-2. Angiogenic factors like VEGF or bFGF did not affect surface detection of CD44v3 suggesting a constitutive expression. The putative functional role for endothelial cell surface CD44v3 was identified in chemotaxis assay when anti-CD44v3 antibody pretreatment proved to be inhibitory for HUVEC. Furthermore, we provided evidence for the CD44v3 protein expression in human endothelial cells in vivo in peritumoral microvessels of both human melanoma and glottic cancers, suggesting a role for this part-time heparan sulfate proteoglycan in tumor induced angiogenesis.     

Vein Involvement During Pancreaticoduodenectomy: Is There a Need for Redefinition of “Borderline Resectable Disease”?

Introduction

Current National Comprehensive Cancer Network guidelines recommend neoadjuvant therapy for borderline resectable pancreatic adenocarcinoma to increase the likelihood of achieving R0 resection. A consensus has not been reached on the degree of venous involvement that constitutes borderline resectability. This study compares the outcome of patients who underwent pancreaticoduodenectomy with or without vein resection without neoadjuvant therapy.

Methods

A multi-institutional database of patients who underwent pancreaticoduodenectomy was reviewed. Patients who required vein resection due to gross vein involvement by tumor were compared to those without evidence of vein involvement.

Results

Of 492 patients undergoing pancreaticoduodenectomy, 70 (14 %) had vein resection and 422 (86 %) did not. There was no difference in R0 resection (66 vs. 75 %, p?=?NS). On multivariate analysis, vein involvement was not predictive of disease-free or overall survival.

Conclusion

This is the largest modern series examining patients with or without isolated vein involvement by pancreas cancer, none of whom received neoadjuvant therapy. Oncological outcome was not different between the two groups. These data suggest that up-front surgical resection is an appropriate option and call into question the inclusion of isolated vein involvement in the definition of “borderline resectable disease.”     

Molecular cloning and regional distribution of rat brain cyclophilin.

Cyclosporine A (CsA) is a potent immunosuppressive drug that has widespread clinical uses in organ transplantation and the treatment of autoimmune disorders. However, the drug's clinical applications are on an empiric basis with a poor understanding of the basic mechanism(s) of action. CsA may exert some of its effects by binding to a cellular receptor protein--the cyclosporine receptor (also called cyclophilin). Cyclophilin (CyP) is an ubiquitous, soluble, cytoplasmic 17 kDa protein which has recently been shown to be a peptide-prolyl isomerase. CsA specifically binds to this protein and inhibits its isomerase activity. A rat cyclophilin cDNA clone was isolated from a rat brain lambda gt11 cDNA library. Northern blot analysis shows a single 1 kb messenger RNA in rat brain. In order to determine the regional distribution of the Cyp mRNA in situ hybridization was performed. The Cyp mRNA appeared to be expressed throughout the brain but there were particularly high levels in the cerebral cortex and hippocampus compared to the relatively low levels in white matter areas and tracts. At the cellular level, the Cyp mRNA is expressed at much higher levels in neurons than in glia. The high levels of Cyp in cortical (neuronal) areas may, in part, explain the global encephalopathic symptoms clinically observed in CsA neurotoxicity.     

Cap formation in a B-lymphocyte cell line is inhibited by pertussis toxin and phorbol ester.

We have examined concanavalin A (Con A)-induced cap formation in a B-lymphocyte derived cell line, LAZ-559. Treatment with pertussis toxin (PT) or phorbol-12-myristate-13-acetate (PMA) prior to exposure of the cells to Con A abolished the capping reaction. The possible role of calcium mobilization was tested using cells pre-loaded with the fluorescent dye Quin2. Both PT and PMA caused inhibition of calcium mobilization at concentrations similar to those observed for the inhibition of capping. The possible identity of the substrate for pertussis toxin was examined by carrying out ADP-ribosylation of the isolated plasma membranes using [alpha-32P]NAD and pertussis toxin. Several bands were observed at molecular weights of 109,000, 43,000, 34,000 and 22,000. Comparative labelling with cholera toxin revealed a separate band at 42,000. The bands at 43,000 and 34,000 are PT specific. Of these, the 43,000 band comigrated with the PT substrate that has been shown to regulate capping in human neutrophils (Lad et al., 1985a, 1986b). PMA-induced phosphorylation was examined in 32P-loaded cells, and multiple bands were observed to be labelled in a dose-dependent manner, at least two of which were very similar in mobility to the PT substrate. Our results suggest that regulation of calcium mobilization and the control of capping via a PMA-sensitive, GTP-binding protein are probably general phenomena observable in multiple cell systems.     

Choroid plexus papilloma of the posterior third ventricle during infancy & childhood: report of two cases with management morbidities

We report two cases of posterior third ventricular choroid plexus papilloma, one in an 8-month-old infant and another in a two-year-old child. These cases presented with features of obstructive hydrocephalus. Both these patients underwent a ventriculo-peritoneal (VP) shunt surgery prior to the tumor excision. Following the VP shunt surgery both patients developed ascitis requiring exteriorization of the abdominal end of the shunt. There was a clear proof of CSF overproduction: 1400-1500 ml/day in the eight-month-old infant and 900-1200 ml/day in the two-year-old child. In the former it was transient and could be treated with revision of the VP shunt whereas in the second case a ventriculo-arterial shunt had to be done. In the second case a staged reduction cranioplasty was also performed for an enormously enlarged head (head circumference--74 cm). Interesting clinical and radiological findings and useful management strategies are described.     

The rate of hydrolysis of phosphomonoester dianions and the exceptional catalytic proficiencies of protein and inositol phosphatases

To evaluate the proficiency of phosphatases as catalysts, the rate of the uncatalyzed hydrolysis of simple phosphate monoester dianions was estimated by extrapolating rates measured over a range of high temperatures. The rate of spontaneous hydrolysis of phenyl phosphate dianion indicates that a linear free energy relationship reported earlier is reliable for leaving groups whose conjugate acids have pKa values up to at least 10. Using Teflon reaction vessels, it proved possible to follow the hydrolysis of methyl phosphate and 3-(4-carboxy)-2,2-dimethylpropyl phosphate in strong alkali. Even in 1 M KOH, the reaction was found to be specific acid catalyzed. These results establish an upper limit for dianion reactivity, which had been overestimated earlier as a result of the leaching by alkali of silicic acid from quartz reaction vessels. The present findings indicate that the half-time for attack by water on alkyl phosphate dianions is 1.1 x 10(12) years (k = 2 x 10(-20) s) at 25 degrees C and that phosphatases involved in cell signaling and regulation produce the largest rate enhancements that have been identified thus far. Protein phosphatase-1 and inositol 1-phosphatase exceed all other known enzymes in their affinities for the altered substrates in the transition state.