Real-world EGFR testing practices for non-small-cell lung cancer by thoracic pathology laboratories across Europe.

BACKGROUND: Testing for epidermal growth factor receptor (EGFR) mutations is an essential recommendation in guidelines for metastatic non-squamous non-small-cell lung cancer, and is considered mandatory in European countries. However, in practice, challenges are often faced when carrying out routine biomarker testing, including access to testing, inadequate tissue samples and long turnaround times (TATs). MATERIALS AND METHODS: To evaluate the real-world EGFR testing practices of European pathology laboratories, an online survey was set up and validated by the Pulmonary Pathology Working Group of the European Society of Pathology and distributed to 64 expert testing laboratories. The retrospective survey focussed on laboratory organisation and daily EGFR testing practice of pathologists and molecular biologists between 2018 and 2021. RESULTS: TATs varied greatly both between and within countries. These discrepancies may be partly due to reflex testing practices, as 20.8% of laboratories carried out EGFR testing only at the request of the clinician. Many laboratories across Europe still favour single-test sequencing as a primary method of EGFR mutation identification; 32.7% indicated that they only used targeted techniques and 45.1% used single-gene testing followed by next-generation sequencing (NGS), depending on the case. Reported testing rates were consistent over time with no significant decrease in the number of EGFR tests carried out in 2020, despite the increased pressure faced by testing facilities during the COVID-19 pandemic. ISO 15189 accreditation was reported by 42.0% of molecular biology laboratories for single-test sequencing, and by 42.3% for NGS. 92.5% of laboratories indicated they regularly participate in an external quality assessment scheme. CONCLUSIONS: These results highlight the strong heterogeneity of EGFR testing that still occurs within thoracic pathology and molecular biology laboratories across Europe. Even among expert testing facilities there is variability in testing capabilities, TAT, reflex testing practice and laboratory accreditation, stressing the need to harmonise reimbursement technologies and decision-making algorithms in Europe.

[Learning curve and short-term clinical outcomes of Mako robotic-assisted direct anterior approach total hip arthroplasty].

Objective: To explore the learning curve and short-term clinical outcomes of Mako robotic-assisted direct anterior approach total hip arthroplasty (THA). Methods: The preoperative basic data, surgical information and postoperative rehabilitation of 50 patients who underwent Mako robotic-assisted THA for hip diseases in Department of Orthopedic Surgery of the 6th People's Hospital Affiliated to Shanghai Jiao Tong University from December 2018 to December 2020 were analyzed retrospectively, included operation time, intraoperative blood loss, postoperative complications, postoperative imaging parameters (abduction angle, anteversion angle, lower limb length difference, eccentricity difference) and postoperative hip joint Harris score (hip Harris score, HHS). There were 16 males and 34 females, with a mean age of 50-79(67±10) years. The postoperative clinical results of Mako robotic-assisted total hip arthroplasty was analyzed. A cumulative sum analysis (CUSUM) was performed on the operation time (OT). The CUSUM learning curve was modeled by curve fitting and R² was used to testify the goodness. The different phase of the learning curve was compared with several observation indicators. Results: All patients were followed up for more than 6 months. Two patients had poor wound healing and 5 patients had symptoms of lateral femoral cutaneous nerve injury, which disappeared within 1-2 months. No serious complications such as dislocation, aseptic loosening, periprosthetic infection or revision occurred in all the patients. The average operation time was (81±16) min, and the intraoperative blood loss was (456±84) ml. The average Harris hip score at the last follow-up was 88.6±2.5. The radiological evaluation showed that the positions of the acetabular cups were all in the Lewinnnek safety zone; the limb length discrepancy was (0.15±0.50) cm, the offset was (-0.11±0.72) cm. The OT decreased with the accumulation of the cases. The CUSUM learning curve was best modeled as cubic curve,the fitting curve reached the top at the 19th case. As a cut-off point, the 19th point divided the learing curve into two phases. There were statistical differences in OT, pelvic array installation time, acetabular registration time, acetabular reaming time (all P<0.05), but there was no significant differences in Harris hip score, acetabular prosthesis anteversion angle and abduction angle between the two groups (all P>0.05). Conclusions: The learning curve of Mako robot-assisted DAA-THA is about 19 cases. Mako robot-assisted DAA-THA can ensure the accuracy of prosthetic placement and the safety of the operation during the learning curve, and the short-term clinical results after surgery is excellent.

Divisional Reconstruction Strategy: The Repair of Perineal Skin Defect After Tumor Resection.

BACKGROUND AND AIMS: The unique anatomical and physiological function of the perineum region makes it difficult to be repaired after tumor resection. We aim to evaluate the efficacy of PSC divisional reconstruction strategy in the reconstruction of perineal skin defect. MATERIALS AND METHODS: This study includes patients undergoing perineal skin defect reconstruction with PSC strategy-P (penis), S (scrotum), C (circum-penoscrotal skin) divisional reconstruction strategy. RESULTS: From August 2013 to August 2018, 47 patients were enrolled in the surgical procedure. The defect area after resection measured 2 cm × 2.5 cm, minimum, and 12 cm × 18 cm, maximum. Among them, the cases involved one, two, and three zones are 12, 10, and 25, respectively. The skin defects were divisionally repaired. All flaps were well survived without complications or scar contracture. No tumor recurrence happened. CONCLUSION: The application of PSC divisional reconstruction strategy is a promising way to repair wounds in circum-penoscrotal skin area. Moreover, this strategy is easy to process and shows no significant complications during follow-up period.

Relationship between electrocardiographic changes and EPO level in stable CAD patients with autonomic nerve functional damage.

OBJECTIVE: To investigate the relationship between electrocardiographic changes and erythropoietin (EPO) level in stable coronary artery disease (CAD) patients with autonomic nerve functional damage. PATIENTS AND METHODS: Clinical data of 96 stable CAD patients who were treated in our hospital from January 2017 to December 2019 were retrospectively analyzed. All patients were grouped according to whether autonomic nerve function damage was combined; the baseline characteristic data and the morphological characteristics of ECG scattergram were compared between 2 groups, and the relationship between ECG scattergram and EPO level & autonomic nerve function was analyzed. RESULTS: The levels of EPO and red cell volume distributing width (RDW) in stable CAD patients with autonomic nerve dysfunction were significantly higher than that of CAD patients without autonomic nerve dysfunction (p<0.05). The length of scattergram in stable CAD patients with autonomic nerve dysfunction was significantly shorter than that of those without autonomic nerve dysfunction (p<0.05). The cometary sign proportion of ECG scattergram in stable CAD patients with autonomic nerve dysfunction was significantly lower than that of stable CAD patients without autonomic nerve dysfunction (p<0.05). There was negative correlation between EPO levels and scattergram length in stable CAD patients with and without autonomic nerve dysfunction (r=0.44, p=0.02). There was no correlation between EPO levels and scatter width in stable CAD patients with and without autonomic nerve dysfunction (r=0.10, p=0.58). The results of binary logistic regression analysis showed that EPO level was the independent risk factor for the occurrence of autonomic dysfunction in patients with stable CAD (p<0.05). The length of scattergram was the independent protective factor of autonomic nerve function impairment in patients with stable CAD (p<0.05). The AUC of EPO level and scattergram was 0.74 and 0.72 respectively, both of which have similar prediction value. CONCLUSIONS: The level of EPO in stable CAD patients with autonomic nerve dysfunction was related to the change of ECG; and the EPO level and scattergram length can be used to predict the occurrence risk of autonomic nerve dysfunction.

[Analysis of differences in epidemiology and clinical features of Guillain-Barré syndrome between rural and urban areas of southern China].

Objective: To explore the differences in epidemiology and clinical features of Guillain- Barré syndrome (GBS) between rural and urban areas of southern China. Methods: The clinical data of 759 hospitalized GBS patients from 31 hospitals of 13 provinces/cities in southern China, between January 1st, 2013 and September 30th, 2016, were collected and analyzed retrospectively. Results: The risk of GBS was higher for males than females in rural and urban areas and the median age was 49 and 48 years, respectively. Seasonal clustering in winter and spring was noted in both rural and urban areas, and the seasonal trend was more markedly in rural areas, but the differences showed no statistical significance. There were 70.37% of patients in rural areas and 73.69% in urban areas who had antecedent respiratory infection. The median time from onset to nadir was 7 days, and Hughes Disability Scale at admission, nadir and discharge were (2.95±1.10 vs 2.84±1.15), (3.25±1.11 vs 3.14±1.21), (2.02±1.24 vs 2.00±1.31) in rural and urban areas respectively. Albuminocytologic dissociation was present in 84.34% of patients in rural areas and 84.62% of cases in urban areas. There were 8.65% and 10.94% of cases in rural and urban areas who required mechanical ventilation during hospitalization, respectively. Demyelinating GBS accounted for 53.29% and 48.77%, respectively, in patients with findings of nerve conduction studies available in rural and urban areas. Conclusions: GBS in rural areas of southern China showed male predominance and a peak of spring and winter occurrence, with respiratory infection as the predominated preceding events and demyelinating GBS being main clinical subtype. Winter and spring showed a higher incidence of GBS in rural and urban areas. There were no significant differences of sex, age, preceding events, season trend, progression of disease, clinical subtypes and cerebrospinal fluid investigations in GBS patients between rural and urban areas.

MiR-29 regulates retinopathy in diabetic mice via the AMPK signaling pathway.

OBJECTIVE: This work aims to study the influence of micro-ribonucleic acid (miR)-29 on the retinopathy in diabetic mice via the adenosine 5'-monophosphate-activated protein kinase (AMPK) signaling pathway. MATERIALS AND METHODS: A total of 24 C57BL/6 mice were randomly divided into normal group (n=12) and model group (n=12). Mice in the normal group were given to normal diet, and those in the model group were prepared for establishing diabetes mouse model. After animal procedures, electroretinogram was performed to detect the latent period and amplitude of b-wave. The expressions of B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X protein (Bax) were detected via immunohistochemistry. The protein levels of the phosphorylated AMPK (p-AMPK) and phosphorylated mammalian target of rapamycin (p-mTOR) were determined using Western blotting. Moreover, miR-29 expression and cell apoptosis were detected via quantitative Polymerase Chain Reaction (qPCR) and terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL), respectively. RESULTS: Compared with those in the normal group, the latent period prolonged and amplitude of b-wave decreased in the model group (p<0.05). Immunohistochemistry showed that compared with normal group, mice in the model group exhibited increased Bax expression and decreased Bcl-2 expression (p<0.05). The Western blotting analysis showed that the protein levels of p-AMPK decreased and p-mTOR increased in the model group compared with those in the normal group (p<0.05). The qPCR revealed that compared with the normal group, the model group had notably decreased miR-29 expression (p<0.05). TUNEL detection displayed that the apoptotic rate was remarkably elevated in the model group compared with that in the normal group (p<0.05). CONCLUSIONS: Inhibition of miR-17-5p up-regulates the expression of VEGF-A and GDNF in MSCs, and promotes the repair of spinal cord injury by MSCs.

Surgical resection versus watchful waiting in low-grade gliomas.

BACKGROUND: Infiltrating low-grade gliomas (LGG; WHO grade 2) typically present with seizures in young adults. LGGs grow continuously and usually transform to higher grade of malignancy, eventually causing progressive disability and premature death. The effect of up-front surgery has been controversial and the impact of molecular biology on the effect of surgery is unknown. We now present long-term results of upfront surgical resection compared with watchful waiting in light of recently established molecular markers. MATERIALS AND METHODS: Population-based parallel cohorts were followed from two Norwegian university hospitals with different surgical treatment strategies and defined geographical catchment regions. In region A watchful waiting was favored while early resection was favored in region B. Thus, the treatment strategy in individual patients depended on their residential address. The inclusion criteria were histopathological diagnosis of supratentorial LGG from 1998 through 2009 in patients 18 years or older. Follow-up ended 1 January 2016. Making regional comparisons, the primary end-point was overall survival. RESULTS: A total of 153 patients (66 from region A, 87 from region B) were included. Early resection was carried out in 19 (29%) patients in region A compared with 75 (86%) patients in region B. Overall survival was 5.8 years (95% CI 4.5-7.2) in region A compared with 14.4 years (95% CI 10.4-18.5) in region B (P < 0.01). The effect of surgical strategy remained after adjustment for molecular markers (P = 0.001). CONCLUSION: In parallel population-based cohorts of LGGs, early surgical resection resulted in a clinical relevant survival benefit. The effect on survival persisted after adjustment for molecular markers.

Investigation of AZF microdeletions in patients with Klinefelter syndrome.

We investigated azoospermia region microdeletions in male infertility patients with Klinefelter syndrome (KFS), as well as the association between azoospermia symptoms in patients with KFS and Y chromosome microdeletion polymorphisms. A total of 111 cases with male infertility confirmed to have KFS (47, XXY) and 94 fertile men were included in this study. Peripheral blood was drawn and DNA was extracted from these samples. Multiplex polymerase chain reaction was performed to screen the partial deletions of 25 sequence-tagged sites on the Y chromosome. In 111 cases with KFS, 1 case contained the AZFb+d+c deletion. The Gr/Gr deletion was identified in 12 KFS cases and 5 control cases. In addition, the b2/b3 deletion was identified in 13 KFS cases and 6 control cases. There were no significant differences in phenotype and genotype of the 2 partial AZFc deletions between patients and controls (P > 0.05). Our results suggest that patients with KFS may also have Y chromosome microdeletions to varying degrees and that the gr/gr deletion and b2/b3 deletion may not play a role in the susceptible genetic background of azoospermia in patients with KFS in the Sichuan population.

BRAF V600E mutation in early-stage multiple myeloma: good response to broad acting drugs and no relation to prognosis.

In this study, we analyzed the prevalence and clone size of BRAF V600E mutation in 209 patients with multiple myeloma and related the results to clinical phenotype, response and survival. Biopsies were screened for BRAF V600E by allele-specific real-time PCR (AS-PCR). Positive results were confirmed by immunohistochemistry, Sanger sequencing and, in three patients from whom we had stored purified myeloma cells, whole-exome sequencing. Eleven patients (5.3%) were BRAF V600E mutation positive by AS-PCR and at least one other method. The fraction of mutated cells varied from 4 to 100%. BRAF V600E-positive patients had no characteristic clinical phenotype except for significantly higher levels of serum creatinine (125 versus 86 µmol/l) Seven of eleven patients responded with at least very good partial response to alkylators, immunomodulatory agents or proteasome inhibitors. Progression-free and overall survival were similar in patients with and without the mutation. By this integrated approach, we found that patients with BRAF V600E mutation responded very well to broad acting drugs and there was no relation to prognosis in early-stage myeloma. In particular, a large mutated cell fraction did not correlate with aggressive disease.

Identification of apple cultivars on the basis of simple sequence repeat markers.

DNA markers are useful tools that play an important role in plant cultivar identification. They are usually based on polymerase chain reaction (PCR) and include simple sequence repeats (SSRs), inter-simple sequence repeats, and random amplified polymorphic DNA. However, DNA markers were not used effectively in the complete identification of plant cultivars because of the lack of known DNA fingerprints. Recently, a novel approach called the cultivar identification diagram (CID) strategy was developed to facilitate the use of DNA markers for separate plant individuals. The CID was designed whereby a polymorphic maker was generated from each PCR that directly allowed for cultivar sample separation at each step. Therefore, it could be used to identify cultivars and varieties easily with fewer primers. In this study, 60 apple cultivars, including a few main cultivars in fields and varieties from descendants (Fuji x Telamon) were examined. Of the 20 pairs of SSR primers screened, 8 pairs gave reproducible, polymorphic DNA amplification patterns. The banding patterns obtained from these 8 primers were used to construct a CID map. Each cultivar or variety in this study was distinguished from the others completely, indicating that this method can be used for efficient cultivar identification. The result contributed to studies on germplasm resources and the seedling industry in fruit trees.

Pedicled lower lid-sharing flap for full-thickness reconstruction of the upper eyelid.

PURPOSE: To explore the clinical effect of the pedicled lower lid-sharing flap for full-thickness reconstruction of the upper eyelid . METHODS: From 2009 to 2013, 13 upper eyelids with meibomian gland carcinoma (13 patients, age range 52-78 years) were excised, and immediately reconstructed with a pedicled lower lid-sharing flap used for full-thickness upper eyelid defects (up to two-thirds of the eyelid width). Traditionally, the flap is divided after 3 to 4 weeks, and the recipient site closed directly. RESULTS: During a 1-18-month follow-up period, no recurrence, lagophthalmos, hypertrophic scar, or bulky appearance was noted in any of the patients. Aesthetic results for the upper eyelid were obtained for all patients. CONCLUSIONS: We conclude that the pedicled lower lid-sharing flap is a safe and reliable method for reconstruction of full-thickness upper eyelid defects. This procedure not only enables eyelid closure for eye protection, but also directly improves the aesthetic appearance of the face. After second-stage surgery, a stable eyelid margin and lashes with good blood supply and an acceptable cosmetic appearance with regard to symmetry of eyelid height, contour, tarsal show, and skin fold were achieved.

Targeted hepatocellular carcinoma proapoptotic BikDD gene therapy.

Hepatocellular carcinoma (HCC), the third leading cause of cancer death in the world, is the most general type of primary liver cancer. Although current treatment modalities, such as liver transplantation, resection, percutaneous ablation, transarterial embolization, chemotherapy and radiotherapy are potentially curative, these methods are not universally applicable to all of HCC patients, especially for those with poor prognosis in which no effective remedy is available. Therefore, development of novel therapeutic approach for the treatment of HCC is urgently needed. In the current study, we developed a promising HCC-targeted gene therapy vector driven by liver cancer-specific α-fetoprotein promoter/enhancer coupled to an established platform technology. The activity of this expression vector is comparable with or even higher than that of strong cytomegalovirus (CMV) promoter and exhibits strong promoter activity in liver cancer cells/tumors, but has nearly no or very low activity in normal cells/organs in vitro and in orthotopic animal models in vivo. Its cancer specificity exceeds that of the CMV promoter, which expresses non-specifically in both normal and tumor cells. In addition, targeted expression of a therapeutic BikDD, a mutant of proapoptotic gene Bik effectively and preferentially killed liver cancer cells, but not normal cells and significantly repressed growth of HCC tumors, and prolonged survival in multiple xenograft and syngeneic orthotopic mouse models of HCC through intravenous systemic gene delivery. Importantly, systemic administration of BikDD by our expression vector exerted no systemically acute toxicity compared with CMV-BikDD in mice. Taken together, this study elucidates a relatively safe and highly effective and specific systemic gene therapy strategy for liver cancer, and is worthy of further development for future clinical trials.

Expression of claudin-3 and claudin-4 in normal, hyperplastic, and malignant endometrial tissue.

To clarify the roles of claudins in endometrial tumorigenesis, we determined levels of protein and messengerRNA (mRNA) expression of claudin-3 and claudin-4 in human endometrial tissue (proliferative phase [PE, n= 25]; secretory phase [SE, n= 25]; simple hyperplasia [SH, n= 20]; complex hyperplasia [CH, n= 12]; atypical hyperplasia [AH, n= 15]; endometrioid adenocarcinoma [EEC, n= 30]) using immunohistochemistry, western blotting, and real-time polymerase chain reaction, respectively. Morphologic changes of tight junctions (TJs) were also observed in normal, hyperplastic, and malignant endometrial tissue. Absence or weak staining for claudin-3 and claudin-4 was observed in PE, SE, SH, and CH, while medium to intense staining was detected in AH and EEC. Staining of claudin-3 and claudin-4 was predominantly localized to the glandular epithelial cell membrane. Expression of claudin-3 and claudin-4 was significantly increased in the groups of AH and EEC in comparison with the groups of CH, SH, and normal cyclic endometrium at both protein and mRNA levels. The highest expression was observed in EEC. Although no relevance was found with regard to FIGO stage and histologic grade, overexpression of claudin-3 and claudin-4, especially claudin-4, significantly correlated with myometrial invasion. Transmission electron microscopy analysis indicated morphologic disruptions of TJs may lag behind the increase of claudins expression. These results demonstrate that claudin-3 and claudin-4 are strongly expressed in AH and EEC, but less frequently in normal endometrium. The upregulation of claudins expression during endometrial carcinogenesis suggests their potential utility as diagnostic and prognostic biomarkers.

Extracorporeal photochemotherapy in patients with cutaneous T-cell lymphoma: is clinical response predictable?

BACKGROUND: Extracorporeal photochemotherapy (ECP) has been accepted as a standard therapy in cutaneous T-cell lymphomas (CTCL), a category of lymphomas mainly resistant to conventional therapies. Approximately one half of patients demonstrate a reduction in skin affliction by at least 50% within 12 months of therapy and are categorized as responders to ECP. Predictive criteria for selecting patients who will respond to ECP are lacking. Such criteria would however, be of great benefit. OBJECTIVES: This study compared T-cell clonality and serum levels of soluble interleukin-2 receptor (sIL-2R), lactate dehydrogenase (LD), neopterin, beta2-microglobulin (beta(2)-M) and granzyme B in CTCL patients in order to evaluate their potential usefulness as predictive markers. PATIENTS/METHODS: Serum and T lymphocytes obtained from 16 patients with CTCL receiving ECP treatment were evaluated in an open retrospective study. RESULTS: We found no evident correlation between detected T-cell clonality and response to ECP. The non-responding group had on average a higher level of serum sIL-2R. This difference was significant after 6 and 12 months of therapy, but not pretreatment. An individual reduction in serum sIL-2R, neopterin and beta(2)-M during a 6-month course of ECP was well correlated to clinical remission. CONCLUSIONS: Seven out of 16 patients were classified as responders. Neither T-cell clonality nor any of the serum markers assessed pretreatment could reliably predict the response to ECP treatment. However, the individual relative changes in sIL-2R, neopterin and beta(2)-M during 6 months of ECP treatment coherently displayed correlation to the clinical response, as assessed after 12 months of ECP treatment.

Increase of intracellular pH in p53-dependent apoptosis of thymocytes induced by gamma radiation.

Irradiation with gamma rays induces apoptosis of thymocytes by a p53-dependent pathway, but its mechanism is not clear. In this study, we report that gamma-ray-induced apoptosis was associated with the intracellular alkalinization of the thymocytes. After exposure to gamma rays, thymocytes underwent apoptosis when cultured in vitro, and the degree of apoptosis was dependent on the incubation period: The longer the incubation period, the greater the number of cells undergoing apoptosis. However, this apoptosis could be inhibited by the acidic condition of the culture. There was a positive correlation between the pHi of thymocytes and the degree of apoptosis. Treatment with gamma radiation induced apoptosis as well as the elevation of the pHi in thymocytes. The intracellular pH was higher in pre-apoptotic thymocytes than in those that did not undergo apoptosis. Furthermore, apoptosis induced by gamma radiation was inhibited by cycloheximide, actinomycin D or the intracellular Ca2+ chelator, TMB-8. The p53 protein is induced after gamma irradiation. Thus it appears that intracellular pH is increased during the gamma-ray-induced p53-dependent apoptosis of thymocytes.

Identification of genetic determinants responsible for the rapid immunosuppressive activity and the low leukemogenic potential of a variant of Friend leukemia virus, FIS-2.

An immunosuppressive variant of Friend murine leukemia virus (F-MuLV), FIS-2, induces suppression of the primary antibody response against sheep erythrocytes (SRBC) in adult NMRI mice more efficiently than the prototype F-MuLV clone 57 (cl.57). It is, however, less potent than F-MuLV cl.57 in inducing erythroleukemia upon inoculation into newborn NMRI mice. Nucleotide sequence analysis shows a high degree of homology between the two viruses. Single point mutations are scattered over both the gag and the env encoding regions. The most notable mutations are the deletion of one direct repeat and a few single point mutations occurring in the binding sites for cellular transcriptional factors in the FIS-2 long terminal repeat region (LTR). To define the genetic determinants responsible for the pathogenic properties of FIS-2, we constructed six chimeras between FIS-2 and F-MuLV cl.57. Adult mice were infected with the chimeras, and their primary antibody responses against SRBC were investigated. The results showed that the fragment encompassing the FIS-2 env encoding region SU is responsible for the increased immunosuppressive activity in adult mice. A leukemogenicity assay was also performed by infecting newborn mice with the chimeras. Consistent with the previous studies, it showed that the deletion of one direct repeat in the FIS-2 LTR is responsible for the long latent period of erythroleukemia induced by FIS-2 in newborn-inoculated mice. However, studies of cell type-specific transcriptional activities of FIS-2 and F-MuLV cl.57 LTRs using LTR-chloramphenicol acetyltransferase constructs showed that the deletion of one direct repeat does not reduce the transcriptional activity of the FIS-2 LTR. The activity is either comparable to or higher than the transcriptional activity of the F-MuLV cl.57 LTR in the different cell lines that we used, even in an erythroleukemia cell line. It seems that the high transcriptional strength of the FIS-2 LTR is not sufficient to give FIS-2 a high leukemogenic effect. This suggestion is inconsistent with the previous suggestion that the transcriptional strength of an LTR in a given cell type is correlated with the leukemogenic potential in the corresponding tissue. In other words, these data indicate that the direct repeats in the F-MuLV LTR may play other roles besides transcriptional enhancer in the leukemogenesis of F-MuLV.

Molecular cloning and characterization of an immunosuppressive and weakly oncogenic variant of Friend murine leukemia virus, FIS-2.

The FIS variant is a weakly leukemogenic, relatively strong immunosuppressive murine retrovirus which was isolated from the T helper cells of adult NMRI mice infected with Friend murine leukemia virus (F-MuLV) complex (FV). Unlike FV, it does not induce acute erythroleukemia but retains the immunosuppressive property of FV and induces suppression of the primary antibody response rapidly and persistently in adult mice. A previous study showed that the FIS variant contains two viral components, a replication-competent virus and a defective virus. In this study, we have biologically purified the FIS variant by end point dilution and we show that the replication-competent virus FIS-2 alone can induce immunosuppression as the parental FIS variant. Most newborn mice infected with FIS-2 developed erythroleukemia, but with an increased latency period compared with that of F-MuLV clone 57. In contrast, FIS-2 induced suppression of the primary antibody response and disease more rapidly than F-MuLV clone 57 in immunocompetent, adult mice. FIS-2 was further molecularly cloned and characterized. Restriction mapping and nucleotide sequence analysis of FIS-2 showed a high degree of homology between FIS-2 and F-MuLV clone 57, suggesting that FIS-2 is a variant of F-MuLV. The striking difference is the deletion of one of the tandem repeats in the FIS-2 long terminal repeat and the single point mutation in the binding sites for core-binding protein and FVa compared with the long terminal repeat of F-MuLV clone 57. Two single point mutations led to the appearance of two extra potential N glycosylation sites in the FIS-2 gag-encoded glycoprotein. Together, the results suggest that FIS-2 represents an interesting murine model to study retrovirus-induced immunosuppression on the basis of its unique combined property of low leukemogenicity and relatively strong and persistent immunosuppressive activity in adult mice.

Synthesis and cytotoxicity studies of novel cyclic peptide-2,6-dimethoxyhydroquinone-3-mercaptoacetic acid conjugates.

In an effort to investigate the use of small-ring-size cyclic peptides as carriers of new antitumor agents, we synthesized three cyclic tripeptide-cytotoxic agent conjugates. The cytotoxic agent conjugated to the epsilon-amino group of the lysyl residue of the cyclic peptides is 2,6-dimethoxyhydroquinone-3-mercaptoacetic acid (DMQ-MA), (Sheh et al., 1992). The cyclic peptides were synthesized by coupling protected amino acid residues in solution and the subsequent cyclization performed by the pentafluorophenyl ester method as described previously (Sheh et al., 1985, 1987, 1990). After deblocking the lysyl-Z group of the peptides, the conjugation was achieved by reaction with the pentafluorophenyl ester of DMQ-MA. The three cyclic peptides exhibited potent cytotoxicity against two solid tumor cell lines (KB and PC-9) under the synergistic activation of L-ascorbic acid. Electron spin resonance (ESR) studies of DMQ-MA and two conjugates showed that massive hydroxyl radicals were generated as a non-linear function of L-ascorbic acid concentration. These studies indicate that the hydroxyl radical is a possible mediator of cytotoxicity for these conjugates and that small-ring-size cyclic peptides are potentially useful carriers of cytotoxic agents.

A low oncogenic variant of Friend murine leukemia virus with strong immunosuppressive properties.

Friend leukemia complex (FLC) is known to induce immunosuppression but the use of FLC in studies of immune cells function is disadvantageous since the immunosuppression always is accompanied by an acute erythroleukemia. To obtain immunosuppressive variants of FLC with reduced leukemogenic potential, we isolated T-helper cells from FLC infected mice, and passed lysates of the cells to recipient uninfected mice. A group of these mice developed a condition distinct from the disease induced by FLC. A viral stock prepared from these mice, designated Fd-MIV for friend derived murine immunodeficiency virus, induced a profound suppression of the primary antibody response without acute transformation in adult NMRI mice. Terminally a wasting disease with weight loss, atrophy of the thymus and lymph nodes and renal disease was observed in some mice. Analysis of viral DNA and RNA from infected NIH 3T3 cells showed that Fd-MIV contained at least two viral components, a 8.4 kb friend murine leukemia virus (F-MuLV) and a 7.4 kb mink cell focus (MCF)/xenotropic virus related genome. The 7.4 kb genome was not detected in Fd-MIV infected, immunocompromised mice indicating that the 8.4 kb genome might be responsible for the disease.