Targeted delivery of human pro-apoptotic enzymes to tumor cells: In vitro studies describing a novel class of recombinant highly cytotoxic agents

The serine protease granzyme B (GrB, 25 kDa) can initiate apoptosis by multiple mechanisms including directly activating caspases, inducing DNA fragmentation, activating the mitochondrial death pathway, and directly cleaving the nuclear matrix. The purpose of this study was to determine whether a recombinant antibody could deliver sufficient amounts of GrB to target cells to generate an apoptotic signal. The gene sequence encoding GrB was attached to the single-chain anti-melanoma antibody scFvMEL (anti-gp240) via a flexible (G(4)S) tether. The 53-kDa GrB/scFvMEL fusion protein was expressed in bacteria and purified by metal affinity chromatography. Western blotting confirmed presence of both scFvMEL and GrB proteins. The fusion construct displayed intact GrB enzymatic activity (specific activity = 2.6 x 10(5) units/ micro mol) similar to native GrB (specific activity = 4.8 x 10(5) units/ micro mol). The construct bound specifically to human A375-M melanoma cells and delivered GrB to the cytosol as assessed by confocal microscopy. Against log-phase melanoma cells, GrB/scFvMEL demonstrated an IC(50) of 20 nM and minimal cytotoxicity to non-target cells at doses of up to 1 micro M. Coadministration of exogenous perforin (PFN) to cells resulted in a slight increase in the cytotoxic effects of the GrB/scFvMEL construct on A375 target cells and a significant increase in cytotoxicity to SKBR3 (non-target) cells. The cytotoxic effects of this fusion construct on target cells were similar to those of the previously described MEL sFv/rGel fusion toxin (IC(50) approximately 20 nM). The construct produced impressive apoptotic effects by 8 h after treatment of target cells. Mediation of the apoptotic effects of GrB/scFvMEL included caspase-3 cleavage and release of cytochrome c into the cytosolic compartment from the mitochondrial compartment. These studies demonstrate that delivery of the human pro-apoptotic pathway enzyme GrB to tumor cells may have significant therapeutic potential for cancer treatment and represents a new class of targeted therapeutic agents with a defined mechanism of action.     

Safety and pharmacokinetics of multiple doses of recombinant human CuZn superoxide dismutase administered intratracheally to premature neonates with respiratory distress syndrome

OBJECTIVES: To examine the safety and pharmacokinetics of multiple intratracheal (IT) doses of recombinant human CuZn superoxide dismutase (rhSOD) in premature infants with respiratory distress syndrome who are at risk for developing bronchopulmonary dysplasia (BPD). Methods. Thirty-three infants (700 to 1300 g) were randomized and blindly received saline, 2.5 mg/kg or 5 mg/kg rhSOD IT within 2 hours of surfactant administration. Infants were treated every 48 hours (as long as endotracheal intubation was required) up to 7 doses. Serial blood and urine studies, chest radiographs, neurosonograms, SOD concentration and activity measurements, and tracheal aspirate (TA) inflammatory markers were assessed throughout the 28-day study. RESULTS: SOD concentrations in serum (0.1 [0.05/0.15] microg/mL-geometric mean with lower/upper confidence intervals), tracheal aspirates (TA) (0.2 [0.1/0.3] microg/mL) and urine (0.3 [0.2/0.4] microg/mL) were similar at baseline in all 3 groups and did not change significantly in the placebo group. In the rhSOD treatment groups, SOD concentrations were increased on day 3 and did not change significantly thereafter over the 14-day dosing period (also measured on days 5, 7, and 13). SOD concentrations averaged 0.4 [0.3/0.5] microg/mL in serum, 0.8 [0.6/1.2] microg/mL in TA and 1.1 [1.0/1.3] microg/mL in urine for the low-dose group and 0.6 [0.5/0.7] microg/mL in serum, 1.1 [0.9/1.5] microg/mL in TA, and 2.2 [1.6/2.9] microg/mL in urine for the high-dose group over the 14-day dosing period. Enzyme activity directly correlated with SOD concentration and rhSOD was active even when excreted in urine. TA markers of acute lung injury (neutrophil chemotactic activity, albumin concentration) were lower in the rhSOD agroups compared with placebo. No significant differences in any clinical outcome variable were noted between groups. CONCLUSIONS: These data indicate that multiple IT doses of rhSOD increase the concentration and activity of the enzyme in serum, TA and urine, reduce TA lung injury markers and are well-tolerated. Further clinical trials examining the efficacy of rhSOD in the prevention of BPD are warranted.     

The impact of glucose-insulin-potassium infusion in acute myocardial infarction on infarct size and left ventricular ejection fraction [ISRCTN56720616]

Background

Stopping antipsychotic treatment can interrupt improvement and exacerbate the illness. The reasons for discontinuing treatment during controlled clinical trials were analyzed to explore this phenomenon.

Methods

A post-hoc, pooled analysis was made of 4 randomized, double-blind clinical trials, 24–28 weeks in duration, involving 1627 patients with schizophrenia or a related disorder. Analyses combined all the atypical antipsychotic treatment groups in the studies.

Results

The majority of patients (53%) stopped their treatment at an early stage. Poor psychiatric response along with worsening symptoms was the most frequently given reason for discontinuing the course (36%), which was substantially more common than discontinuation due to poor tolerability of the medication (12%). This phenomenon was corroborated by less improvement in patients who discontinued treatment compared with those who completed, based on the PANSS total scores. Discontinuation due to poor response was, apparently, more predominantly linked to patient perception than to physicians' conclusions alone (80% vs. 20%). Discontinuation due to patient perception of poor response appeared to occur particularly early in the course of treatment. Patients who discontinued due to poor toleration of the medication responded in a more comparable manner with completers.

Conclusion

Discontinuing treatment may lead to exacerbation of symptoms, undermining therapeutic progress. In these studies, poor response to treatment and worsening of underlying psychiatric symptoms, and to a lesser extent, intolerability to medication were the primary contributors to treatment being discontinued. Our findings suggest that adherence may be enhanced by effective symptom control, as objectively measured and as subjectively perceived. Such strategies may improve patients' willingness to undertake long-term therapy and increase the likelihood of a better prognosis.     

Evaluation of whole genome amplification protocols for array and oligonucleotide CGH.

Genome-based technologies such as genomic arrays and next generation sequencing are poised to make significant contributions to clinical oncology. However, translation of these technologies to the clinic will require that they produce high-quality reproducible data from small archived tumor specimens and biopsies. Herein, we report on a systematic and comprehensive microarray analysis comparing multiple whole genome amplification methods using a variety of difficult clinical specimens, including formalin-fixed and paraffin-embedded tissues. Quantitative analysis and clustering suggest that Sigma's whole genome amplification protocol performed best on all specimens and, moreover, worked well with a formalin-fixed, paraffin-embedded biopsy.     

Predicting cancer development in oral leukoplakia: ten years of translational research.

Our 10-year translational study of the oral premalignant lesion (OPL) model has advanced the basic understanding of carcinogenesis. Although retinoids have established activity in this model, a substantial percentage of our OPL patients progress to cancer, especially after treatment is stopped. On the basis of our 10-year OPL study, we have developed the first comprehensive tool for assessing cancer risk of OPL patients. This cancer risk assessment tool incorporates medical/demographic variables, epidemiological factors, and cellular and molecular biomarkers. Between 1988 and 1991, 70 advanced OPL patients were enrolled in a chemoprevention trial of induction with high dose isotretinoin (1.5 mg/kg/day for 3 months) followed by 9 months of maintenance treatment with either low dose isotretinoin (0.5 mg/kg/day) or beta-carotene (30 mg/d; total treatment duration, 1 year). We assessed the relationship between cancer risk factors and time to cancer development by means of exploratory data analysis, logrank test, Cox proportional hazard model, and recursive partitioning. With a median follow-up of 7 years, 22 of our 70 patients (31.4%) developed cancers in the upper aerodigestive tract following treatment. The overall cancer incidence was 5.7% per year. The most predictive factors of cancer risk are OPL histology, cancer history, and three of the five biomarkers we assessed (chromosomal polysomy, p53 protein expression, and loss of heterozygosity at chromosome 3p or 9p). In the multivariable Cox model, histology (P = 0.0003) and the combined biomarker score of chromosomal polysomy, p53, and loss of heterozygosity (P = 0.0008) are the strongest predictors for cancer development. Retinoic acid receptor beta and micronuclei were not associated with increased cancer risk. We have demonstrated a successful strategy of comprehensive cancer risk assessment in OPL patients. Combining conventional medical/demographic variables and a panel of three biomarkers can identify high risk patients in our sample. This result will need to be validated by future studies. With the identification of high risk individuals, more efficient chemoprevention trials and molecular targeting studies can be designed.     

Human malignancy-associated nucleolar antigen as a marker for tumor cells in patients with acute leukemia

Tumor burden in adult patients with acute leukemia is assessed using the percentage of blast cells in the bone marrow or blood. It is clear, however, that not all blast cells are leukemic cells, especially during rapid marrow regeneration. Similarly, some leukemia cell lines have been shown to differentiate in vitro, and the same process also occurs in vivo. Therefore, the leukemic burden may be due to more differentiated cells as well as to blast cells. The purpose of this study was to investigate whether the human malignancy-associated nucleolar antigen (HMNA) could be used as a marker for leukemic cells and to examine its potential as a diagnostic tool. The proportion of HMNA-positive cells in the bone marrow of patients with acute leukemia was determined by indirect immunofluorescence with antibodies to HMNA and was compared with the differential counts routinely made in the clinic laboratory. The percentages of HMNA-positive cells among the nucleated cells in the marrow of 72 patients with clinical evidence of leukemia were significantly higher (range 9%-98%, median 83%) than those observed for nonleukemic individuals (range less than 0.05%-2.5%, median 1%) or for fractions of marrow cells from normal volunteers enriched for normal early progenitor cells (less than or equal to 2%). Patients with leukemia in remission had a lower percentage of HMNA- positive cells (range 0%-83%, median 3%). The percentage of HMNA- positive cells increased as patients approached relapse. Although the percentage of HMNA-positive cells was related to the percentage of blast cells in the bone marrow of the patients with leukemia, some partially differentiated cells were also HMNA-positive in some specimens, and some blastic cells were HMNA-negative in other specimens. These studies indicate the potential usefulness of HMNA as a marker for leukemic cells.     

Testicular tumors: prospective analysis of real-time US patterns and abdominal staging

Echomorphologic features of 57 testicular lesions (48 neoplasms, nine benign lesions) were analyzed prospectively by means of high-resolution real-time ultrasonography (US). There was a broad spectrum of texture patterns for testicular malignancies, 92% of which exhibited a predominantly decreased echogenicity compared with that of normal tissue. US findings alone could not be used to make a specific prediction of malignant disease or to classify a neoplasm histologically. However, different kinds of tumors exhibited characteristic echomorphologic features that corresponded to their gross morphologic appearance; a knowledge of these echomorphologic features is extremely useful for the US differentiation of tumors from nonneoplastic scrotal pathologic conditions. All neoplasms in this series displayed distinct abnormalities in parenchymal texture, and all were detected with US. The sensitivity and predictive value of a normal sonogram were as high as 100%. With regard to the detection of retroperitoneal lymph node metastases from testicular tumors, abdominal US had an overall accuracy rate of 96% and was of superior diagnostic value compared with bipedal lymphangiography.     

MR imaging of the nasopharynx and floor of the middle cranial fossa. Part I. Normal anatomy

The normal anatomy of the nasopharynx and floor of the middle cranial fossa was analyzed with magnetic resonance (MR) imaging. MR images from five healthy volunteers were correlated with whole-organ cryomicrotome sections from three cadavers. Anatomic connections exist between the paranasopharyngeal spaces and the surface structures of the skull base. These anatomic connections include the intimate relationship between the eustachian tube and the pharyngobasilar fascia, the attachment of the muscles of mastication and deglutition to the skull base, and vascular and nervous structures in the foramina. The inherent contrast between the soft tissues of the nasopharynx and related structures and the bone of the floor of the middle cranial fossa allowed excellent visualization of these anatomic connections.     

Update of staging and risk assessment for prostate cancer patients

PURPOSE OF REVIEW: This paper will review the current staging system for prostate adenocarcinoma patients, and will also review new information that can be combined with clinical and pathological staging in order to assess a patient's risk of success or failure of treatment. RECENT FINDINGS: There has been significant stage migration of prostate cancer patients in the past 15 years, such that patients are currently being diagnosed younger, with lower clinical stages and serum prostate-specific antigen levels, and a lower risk of metastatic disease than previously. The incorporation of the results of extended prostate biopsy schemes, with stage, grade and serum prostate-specific antigen levels, improves the risk assessment of newly diagnosed prostate cancer patients. New imaging techniques, such as transrectal ultrasound Doppler flow and magnetic resonance spectroscopy hold promise for improving risk assessment. Molecular biomarkers may improve risk assessment in the future, although none are currently approved by the US Food and Drug Administration for this indication. Gene chip arrays may further refine risk assessment and assist with the identification of therapeutic targets. SUMMARY: There has been significant stage migration of prostate cancer patients in the prostate-specific antigen era. Incorporating biopsy information into nomograms and risk assessment equations improves upon clinical staging and risk assessment. New imaging techniques, molecular markers and gene chip arrays hold promise for future risk assessment.