The yeast two-hybrid system reveals no interaction between p73α and SV40 large T-antigen

Summary.  SV40 and/ or DNA sequences indistinguishable from SV40 have been detected in several types of human tumours. The oncoprotein of Simian virus 40, SV40 large T-antigen (Tag), is known to bind and inactivate tumour suppressor proteins, such as members of the retinoblastoma family and p53, thereby promoting cell transformation. In this study, we used the yeast two-hybrid system to investigate whether the Simian virus 40 (SV40) large T-antigen is able to interact with p73, a noval discovered putative tumour suppressor, that is homologous both structurally and functionally to p53. The yeast two-hybrid system is a genetic method to detect protein-protein-interactions in vivo. Our results suggest that the SV40 large T-antigen is not able to bind p73 in yeast although both proteins are expressed in the transformed yeast strain as was shown by western blot analysis. Received May 20, 1998 Accepted September 28, 1998     

Expression of CD40/CD40 ligand and Bcl-2 family proteins in labial salivary glands of patients with Sjögren's syndrome

Lymphocytes infiltrating the salivary glands of patients with Sj?gren's syndrome (SS) are activated and resist apoptosis. We determined the role of interactions between CD40 and CD40 ligand (CD40L) in these infiltrating lymphocytes on B-cell differentiation and expression of Bcl-2 family proteins. Ten human T-cell leukemia/lymphoma virus-I (HTLV-I)-seronegative and eight HTLV-I-seropositive SS patients were examined in the present study. Immunohistochemistry was performed to examine the expression of CD3, CD20, PCA-1, CD40, CD40L, Bcl-2, Bax, and Bcl-x on T and B lymphocytes infiltrating labial salivary glands of SS patients. We also examined the expression of CD40 and CD40L on peripheral blood lymphocytes of the same patients by using flow cytometry. CD40L was not expressed on peripheral blood lymphocytes of SS patients. Peripheral blood B cells but not T cells expressed CD40. In contrast, >50% of mononuclear cells, including T and B cells infiltrating the glands, expressed CD40. In addition, a clear expression of CD40L in both infiltrating T cells and B cells, and that of PCA-1, was also demonstrated. Surprisingly, the expression of Bcl-2 and Bcl-x was colocalized with that of CD40 determined by mirror section technique. Bcl-x was also abundantly expressed on infiltrating mononuclear cells, but, Bax expression was relatively less than that of Bcl-2 or Bcl-x. The expression of the above molecules was not different between HTLV-I-seronegative and HTLV-I-seropositive SS patients. Our results indicate that CD40/CD40L pathways could be augmented in salivary glands of SS patients, inducing B-cell differentiation to PCA-1 + plasma cells. Immunohistochemical analysis also suggests that signaling through CD40 by means of CD40L increases the expression of Bcl-2 as well as Bcl-x in infiltrating lymphocytes, providing the resistance against apoptosis. Our findings were commonly observed in SS patients irrespective of HTLV-I seropositivity.     

A CD40–CD95L fusion protein interferes with CD40L-induced prosurvival signaling and allows membrane CD40L-restricted activation of CD95

We analyzed a novel bifunctional fusion protein, CD40ed–CD95Led, consisting amino-terminally of the extracellular domain of CD40 and carboxy-terminally of the extracellular domain of CD95L. On cells lacking CD40L, this fusion protein is poorly active with respect to CD95 activation [median effective dose (ED50)>1 μg/ml], but it stimulates CD95 signaling with high efficiency upon binding to membrane-expressed CD40L (ED50<1 ng/ml). Thus, cell surface immobilization mediated by the CD40 part of the molecule unmasks the high-latent, CD95-stimulating capacity of the otherwise poorly active CD95L fusion protein. Moreover, interaction of the CD40 part of CD40ed–CD95Led with CD40L prevents the activation of cellular CD40. The CD40ed–CD95Led fusion protein therefore simultaneously blocks antiapoptotic CD40 activation and induces CD95-mediated apoptosis. Indeed, T47D cells displaying an antiapoptotic autocrine CD40–CD40L signaling loop were significantly more sensitive toward CD40ed–CD95Led than toward soluble CD95L artificially activated by crosslinking. Fusion proteins of RANK and CD95L (RANKed–CD95Led) and CD40 and tumor necrosis factor-related apoptosis inducing ligand (TRAIL) (CD40ed–TRAILed), with domain architectures similar to CD40ed–Cd95Led, displayed RANKL-dependent CD95 and CD40L-dependent TRAILR2 activation, respectively, indicating the principle feasibility of this fusion protein design.Klaus Pfizenmaier and Harald Wajant contributed equally to this work.     

Alirocumab dosing patterns during 40 months of open-label treatment in patients with heterozygous familial hypercholesterolemia

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Do 40% of patients resected for barrett esophagus with high-grade dysplasia have unsuspected adenocarcinoma?

Results of studies conducted in the last 2 decades suggest that the detection of high-grade dysplasia in patients with Barrett esophagus is the harbinger of a synchronous adenocarcinoma, which remains undetected even by rigorous biopsy protocols but is discovered during resection of the esophagus. The reported prevalence of synchronous carcinomas ranges from 0% to 75%. Other researchers maintain that appropriate surveillance programs can be used to detect carcinomas at a curable stage and to prevent unnecessary esophagectomies. Both logistical difficulties and potential methodological pitfalls have plagued many studies designed to investigate this issue. A large multicenter study that would stratify participants for hitherto unexplored variables (eg, age, gender, and ethnic background) may be required before the 40% occult cancer prevalence can be either confirmed or refuted. However, the large scale needed for such a study to provide reliable data and new developments in endoscopic imaging (eg, magnification endoscopy and optical coherence tomography) and endoscopic therapy (eg, mucosectomy) are likely to make such a study both ethically unacceptable and logistically and financially unfeasible. Future research should utilize the combination of new endoscopic technologies with the continuing search for validated biomarkers that help predict the biological behavior of Barrett epithelium in individual patients, with a particular focus on the possible development of preneoplastic and neoplastic lesions. Pathologists who chose to shift their focus from the traditional morphological investigation of dysplasia to the search for usable biomarkers can position themselves at the center of innovative research projects that could radically modify the management of patients with Barrett esophagus.     

Effect of CD40/CD40L signaling on IL-10-producing regulatory B cells in Chinese children with Henoch-Schönlein purpura nephritis

The aim of the present study was to examine the role and mechanism of interleukin-10 (IL-10)-producing regulatory B cells (B10 cells) in the pathogenesis of Henoch-Schönlein purpura nephritis (HSPN). We examined the percentage of B10 cells, CD19⁺CD24^hiCD38^hi B cells, CD19⁺CD24^hiCD27⁺ B cells, Th17 cells, and T regulatory (Treg) cells within the peripheral blood mononuclear cell (PBMC) population in healthy subjects and HSP/HSPN patients. The percentage of B10 cells and CD19⁺CD24^hiCD38^hi B cells was reduced in HSPN patients and that of CD19⁺CD24^hiCD27⁺ B cells was decreased only in HSPN patients with hematuria and proteinuria or massive proteinuria. The expression of IL-10 by B10 cells and their subsets was decreased in HSPN patients and returned to normal levels in HSP/HSPN patients in remission. B10 cells and their subsets negatively correlated with the Th17/Treg ratio. There was no difference in B10pro + B10 cells, Th17 cells, Treg cells, and the Th17/Treg ratio between children with HSP/HSPN and healthy controls after CD40L stimulation. On the other hand, the level of IL-10 expressed by CD19⁺CD40⁺ B cells was decreased in HSPN, and the percentage of B10pro + B10 cells and Treg cells was reduced and that of Th17 cell was increased in the presence of anti-CD40L monoclonal antibody (mAb). Thus, decreased B10 cells and CD19⁺CD24^hiCD38^hi B cells may function as an early marker of renal impairment in HSPN. The dysfunction of B10 cells may play a role in the pathogenesis of HSPN by regulating the Th17/Treg balance. Moreover, the CD40/CD40L signaling pathway may play a role in B10 cell differentiation and functional maturation.     

Do cycle disturbances explain the age-related decline of female fertility? Cycle characteristics of women aged over 40 years compared with a reference population of young women

BACKGROUND: The cause of declining fertility with age, in women who still have regular menstrual cycles, is not clear. METHODS: Follicle development, endometrial growth and hormonal patterns were evaluated in cycles of older women (aged 41-46 years; n = 26) who previously were normally fertile, and these cycles were compared with a reference group of relatively young fertile women (aged 22-34 years; n = 35). RESULTS: Clearly abnormal cycles were found in only two women in the older age group, and in one woman in the younger group. The main differences between the age groups were a shorter follicular phase and cycle length in the older group, in combination with higher FSH levels in the late luteal and early follicular phase. In contrast to published data which suggest an "accelerated" follicle development in older women, sonographical and hormonal evidence was found of an "advanced" follicle growth, with an earlier start already during the luteal phase of the preceding cycle, and an advanced selection and ovulation of the dominant follicle. CONCLUSIONS: Such an earlier start of follicle growth in a possibly less favourable hormonal environment, as well as a limited oocyte pool, may contribute to a decreased follicle and oocyte quality, resulting in diminished fertility in ageing women.     

Ovulation induction combined with intrauterine insemination in women 40 years of age and older: is it worthwhile?

The use of ovulation induction combined with intrauterine insemination(IUI) as a treatment for subfertility in women with patent Fallopiantubes has increased in recent years. Little is known regardingthe efficacy of this treatment in women aged40 years. We reviewedour data in our ovulation induction with IUI programme for 168consecutive patients aged 40 years undergoing a total of 469cycles of treatment. Either sequential clomiphene citrate andhuman menopausal gonadotrophins or daily gonadotrophins wereutilized along with timed IUI insemination. In 402 completedcycles, 28 clinical pregnancies occurred. The pregnancy lossrate was 34.4%. The overall ongoing/viable pregnancy rates perinitiated and completed cycles were 4.47 and 5.22% respectively.No viable pregnancies occurred in 136 cycles in women aged 43years. The ongoing/viable cycle fecundity rates for women aged40, 41, and 42 years were 9.6, 5.2, and 2.4% percycle respectively.When utilized in women aged40 years, ovulation induction withIUI is most likely to result in successful pregnancy in women40–42 years of age. Women43 years should consider otheralternatives such as adoption or egg donation.     

Expression of the L-type calcium channel with two different β subunits and its modulation by Ro 40-5967

The smooth muscle 1_Cb subunit of the L-type calcium channel was expressed alone (CHO ₁ cell) or together with the skeletal 1 (CHO1 cell) subunit or smooth muscle 3 (CHO ₁ 3 cell) subunit in Chinese hamster ovary (CHO) cells. The interaction of the expressed calcium channels with the non-dihydropyridine calcium channel blocker Ro 40-5967 was studied. Ro 40-5967 decreased isradipine binding by an apparent allosteric interaction and blocked the barium inward currents (I _Ba) in a voltage- and use-dependent manner in all cells. The steady-state inactivation curves were shifted to hyperpolarizing potentials in the presence of Ro 40-5967. The rate of channel inactivation was increased in CHO ₁ and CHO ₁ 3 cells. The shift in the steadystate inactivation curve and the increase in channel inactivation were less pronounced in CHO ₁ 1 cells than in the other cell lines. Low concentrations of Ro 40-5967 increased I _Ba by up to 198% in 33% of the CHO ₁ 1 cells. In addition, higher concentrations of Ro 40-5967 were required to inhibit I _Ba in 60% of the CHO ₁ 3 cells. These results suggest that the subunits modify the interaction of the non-dihydropyridine Ro 40-5967 with the expressed calcium channel ₁ subunit.Dedicated to the late Professor Dr. W. Osterrieder     

AdCD40L--crossing the valley of death?

CD40-mediated cancer therapy has been under development since it became clear that CD40 plays a profound role in the stimulation of adaptive immune responses. Further, CD40 signaling on tumor cells may lead to growth arrest or even apoptosis that improves therapy outcome. The therapeutic window is appealing since the immune system is selective and normal cells do not apoptose upon CD40 signaling. AdCD40L is an adenoviral-based immunostimulatory gene therapy under evaluation for its efficacy to treat cancer. Because of its nature, the adenoviral backbone will stimulate TLRs while CD40L potentiates the shifts toward Th1 type of immunity. AdCD40L has shown efficacy in various murine models, and safety studies have been performed on dog patients and in human clinical trials. AdCD40L has been used for both ex vivo gene modification of tumor cell vaccines as well as for direct intratumoral injections. Lately, an oncolytic vector has been used to further increase the eradication of solid tumors that as a consequence further boosts the release of tumor antigens and creates danger signaling in the tumor micro milieu. This review discusses the currently unfolding mechanisms of action of AdCD40L gene therapy and its possibilities to reach clinical care.     

Clinical Follow-Up of 11 Argentinian CD40L-Deficient Patients with 7 Unique Mutations Including the So-Called “Milder” Mutants

CD40 ligand (CD40L) deficiency is an X-linked combined immunodeficiency characterized by impaired class switch recombination. We analyzed clinical and molecular findings in 11 Argentinian patients from seven unrelated families. The mean age at onset of symptoms was 1.1 years (0.5-3.0 years) and the 10 alive patients have a median age of 17 years. We identified two nonsense mutations, including R11X reported as a "hypomorphic" defect, four missense mutations, and one point deletion. Although R11X was associated herein with parvovirus B19-anemia and higher Igs levels as previously described, histoplasmosis and Pneumocystis jiroveci pneumonia were also present. Other so-called "milder" mutation, T254M, was present in three related patients clinically and immunologically undistinguishable from the rest of the cohort. Furthermore, 10 of the 11 patients, having heterogeneous mutations, never had persistent neutropenia, none presented Cryptosporidium sp. infection nor developed liver-biliary tract disease, highlighting the debatable concept of "milder" mutations.     

Severe congenital neutropenia or hyper-IgM syndrome? A novel mutation of CD40 ligand in a patient with severe neutropenia

Severe congenital neutropenia (SCN) and CD40 ligand deficiency (CD40LD) are two primary immunodeficiency diseases caused by different underlying genetic defects. In this report, we present a case who clinically presented as a SCN patient, but subsequent mutation analysis of this patient was compatible with CD40LD. The patient is a 3-year-old boy, who was referred to our center because of pneumonia, oral and anal ulcers, and periodontitis. As severe consistent neutropenia and maturation arrest in the myeloid series were observed in the bone marrow, a diagnosis of SCN was made. However, no mutations were found in the ELA2 and HAX1 genes. As functional T cell defects were observed, we suspected CD40LD. DNA sequencing showed a 17-base pair deletion in the CD40L gene. Although the patient did not have a decreased serum level of IgA, and his serum IgM level was within the normal range, the diagnosis of CD40LD was confirmed, suggesting that CD40LD should be suspected in any male patient with recurrent infections and neutropenia.     

Correlation of clinical outcomes with β-glucan levels in patients with invasive candidiasis

The correlation of β-glucan (BG) levels with clinical outcomes in invasive candidiasis (IC) remains unknown. Patients with proven IC were followed prospectively from diagnosis to outcome with twice-weekly serum BG sampling. Correlation of BG with clinical outcome was assessed in each patient. BG levels tend to decrease in successfully treated patients and increase in treatment failures. BG levels may be useful as surrogates for outcome evaluation of IC.     

Nuclear translocation of β-catenin synchronized with loss of E-cadherin in oral epithelial dysplasia with a characteristic two-phase appearance

Alvarado C G, Maruyama S, Cheng J, Ida‐Yonemochi H, Kobayashi T, Yamazaki M, Takagi R & Saku T
(2011) Histopathology 59 , 283–291 Nuclear translocation of β‐catenin synchronized with loss of E‐cadherin in oral epithelial dysplasia with a characteristic two‐phase appearance Aims: One of the important histopathological characteristics of oral epithelial dysplasia is a two‐phase appearance of rete processes, comprising an upper layer of keratinized cells and a lower layer of basaloid cells, and thereby creating a sharp contrast between these two separate cell populations. The aim of this study was to determine the cellular adhesion status of the basaloid cells. Methods and results: Immunohistochemistry for β‐catenin, E‐cadherin and their related molecules was carried out in surgical specimens of two‐phase epithelial dysplasia of the oral mucosa. The lower‐half basaloid cells and the upper keratinized cells were microdissected separately, and extracted DNA samples were subjected to methylation‐specific polymerase chain reaction amplification for E‐cadherin. β‐Catenin was immunolocalized within the nuclei and cytoplasm of Ki67‐positive lower‐half basaloid cells, as well as on the cell membrane of upper parakeratotic cells. The basaloid cells of the lower‐half were also positive for matrix metalloproteinase‐7 and cyclin D1, β‐catenin target gene products, α‐dystroglycan, tenascin‐C, and perlecan, but not for E‐cadherin. The promoter region of the E‐cadherin gene was hypermethylated. Conclusions: The solid proliferation of lower‐half E‐cadherin‐free basaloid cells is enhanced by Wnt signalling cascades, as well as by the intraepithelial extracellular matrix or its bound growth factors.     

Pseudoangiomatous stromal hyperplasia (PASH) of the breast with foci of morphologic malignancy: a case of PASH with malignant transformation?

Pseudoangiomatous stromal hyperplasia (PASH) is a benign proliferation of the hormonally responsive, specialized mammary stroma characterized by slit-like pseudovascular spaces lined by bland spindle cells. It is usually an incidental microscopic finding but in some cases it may present as a slowly growing mass. A malignant counterpart for this lesion has not been reported. We describe a case of PASH with foci of malignant histologic features presenting as a slowly growing mass in a 30-year-old woman. The previously reported variants of PASH and the other mammary stromal lesions related to PASH are also discussed. This is perhaps the first case of PASH with foci of malignant histologic features reported in the literature and represents a rare sarcoma derived from specialized hormonally responsive mammary stroma.     

Modulation of the humoral immune response by targeting CD40 and FcγRII/III; delivery of soluble but not particulate antigen to CD40 enhances antibody responses with a Th1 bias

Targeted delivery of antigen improves immunogenicity and can obviate the use of adjuvants. In addition to molecular targeting based on affinity interactions, particle-based antigen targeting to myeloid cells is also an efficient means to enhance immune responses. We compared the efficiency of targeting a model antigen, streptavidin, to CD40 and low affinity Fc gamma receptors II and III, either in a soluble or in a particulate form. Single chain fragments targeting these receptors were used to generate soluble tetramers with streptavidin or to decorate streptavidin coated nanobeads, and mice were immunized with the different formulations. Whereas particulate presentation of streptavidin enhanced total IgG1 and IgG2a levels, overall antigen specific antibody production increased in the case of targeted soluble antigen only, as assessed by reverse protein arrays and ELISPOT. In particular, soluble CD40 targeted antigen induced the strongest IgG2a responses, suggesting a Th1 bias compared to FcgammaRII/III targeting. Combined targeting to these receptors did not further increase immunogenicity. Thus, in our model, affinity targeting of soluble antigen to CD40 proved to be superior to particle-mediated delivery both in terms of antibody quantity and quality.     

Changes in the oncolytic properties of the serum of rats with Guérin's carcinoma undergoing treatment with sarcolysin

Summary An attempt was made to employ the oncolytic reaction to evaluate the efficacy of sarcolysin treatment in Guérin's rat carcinoma. The oncolytic properties of the sera were periodically investigated in rats suffering from this tumor and treated with sarcolysin. The results obtained were compared with the changes in the oncolytic properties of the serum of animals with tumors untreated with this perparation. It was demonstrated that the rise of the serum oncolytic activity in the treated animals is associated with the reduction of the tumor size. Conversely, the activity fall in the serum oncolytic activity in rats suffering from tumors not treated with sarcolysin is associated with the continuous growth of the tumor. It appears that determination of the oncological properties of the serum during sarcolysin treatment of animals suffering from tumors would enable the efficacy of this therapy on the tumor to be evaluated.Presented by Active Member AMN SSSR N. N. Zhukov-Verezhnikov