Myxoma virus lacking the host range determinant M062 stimulates cGAS-dependent type 1 interferon response and unique transcriptomic changes in human monocytes/macrophages.

The evolutionarily successful poxviruses possess effective and diverse strategies to circumvent or overcome host defense mechanisms. Poxviruses encode many immunoregulatory proteins to evade host immunity to establish a productive infection and have unique means of inhibiting DNA sensing-dependent type 1 interferon (IFN-I) responses, a necessity given their dsDNA genome and exclusively cytoplasmic life cycle. We found that the key DNA sensing inhibition by poxvirus infection was dominant during the early stage of poxvirus infection before DNA replication. In an effort to identify the poxvirus gene products which subdue the antiviral proinflammatory responses (e.g., IFN-I response), we investigated the function of one early gene that is the known host range determinant from the highly conserved poxvirus host range C7L superfamily, myxoma virus (MYXV) M062. Host range factors are unique features of poxviruses that determine the species and cell type tropism. Almost all sequenced mammalian poxviruses retain at least one homologue of the poxvirus host range C7L superfamily. In MYXV, a rabbit-specific poxvirus, the dominant and broad-spectrum host range determinant of the C7L superfamily is the M062R gene. The M062R gene product is essential for MYXV infection in almost all cells tested from different mammalian species and specifically inhibits the function of host Sterile α Motif Domain-containing 9 (SAMD9), as M062R-null (ΔM062R) MYXV causes abortive infection in a SAMD9-dependent manner. In this study we investigated the immunostimulatory property of the ΔM062R. We found that the replication-defective ΔM062R activated host DNA sensing pathway during infection in a cGAS-dependent fashion and that knocking down SAMD9 expression attenuated proinflammatory responses. Moreover, transcriptomic analyses showed a unique feature of the host gene expression landscape that is different from the dsDNA alone-stimulated inflammatory state. This study establishes a link between the anti-neoplastic function of SAMD9 and the regulation of innate immune responses.

Characterization of Morreton virus as an oncolytic virotherapy platform for liver cancers.

BACKGROUND: Morreton virus (MORV) is an oncolytic Vesiculovirus , genetically distinct from vesicular stomatitis virus (VSV). AIM: To report that MORV induced potent cytopathic effects (CPEs) in cholangiocarcinoma (CCA) and hepatocellular carcinoma (HCC) in vitro models. APPROACH AND RESULTS: In preliminary safety analyses, high intranasal doses (up to 10 10 50% tissue culture infectious dose [TCID 50 ]) of MORV were not associated with significant adverse effects in immune competent, non-tumor-bearing mice. MORV was shown to be efficacious in a Hep3B hepatocellular cancer xenograft model but not in a CCA xenograft HuCCT1 model. In an immune competent, syngeneic murine CCA model, single intratumoral treatments with MORV (1 × 10 7 TCID 50 ) triggered a robust antitumor immune response leading to substantial tumor regression and disease control at a dose 10-fold lower than VSV (1 × 10 8 TCID 50 ). MORV led to increased CD8 + cytotoxic T cells without compensatory increases in tumor-associated macrophages and granulocytic or monocytic myeloid-derived suppressor cells. CONCLUSIONS: Our findings indicate that wild-type MORV is safe and can induce potent tumor regression via immune-mediated and immune-independent mechanisms in HCC and CCA animal models without dose limiting adverse events. These data warrant further development and clinical translation of MORV as an oncolytic virotherapy platform.

The glycosyltransferase GnT-III activates Notch signaling and drives stem cell expansion to promote the growth and invasion of ovarian cancer.

Glycosylation changes associated with cellular transformation can facilitate the growth and progression of tumors. Previously we discovered that the gene Mgat3 encoding the glycosyltransferase GnT-III is elevated in epithelial ovarian carcinomas (EOCs) and leads to the production of abnormal truncated N-linked glycan structures instead of the typical bisected forms. In this study, we are interested in discovering how these abnormal glycans impact the growth and progression of ovarian cancer. We have discovered using stable shRNA gene suppression that GnT-III expression controls the expansion of side-population cells, also known as cancer stem cells. More specifically, we found that GnT-III expression regulates the levels and activation of the heavily glycosylated Notch receptor involved in normal and malignant development. Suppression of GnT-III in EOC cell lines and primary tumor-derived cells resulted in an inhibition of Notch signaling that was more potent than pharmacologic blockage of Notch activation via γ-secretase inhibition. The inhibition resulted from the redirection of the Notch receptor to the lysosome, a novel mechanism. These findings demonstrate a new role for bisecting glycosylation in the control of Notch transport and demonstrate the therapeutic potential of inhibiting GnT-III as a treatment for controlling EOC growth and recurrence.

A comparison of alum sludge with peat for aqueous glyphosate removal for maximizing their value for practical use.

This study compares and contrasts the glyphosate removal efficiency of alum sludge (waterworks residue) and Irish peat in aqueous solution. Organic phosphonate of glyphosate aqueous solution was removed in pot tests separately filled with peat and alum sludge, while effluent samples were taken from each pot to analyse the concentration of phosphorus (P) and COD (chemical oxygen demand); physical and chemical analysis for both media before and after use was carried out subsequently. The results show that the P removal capacity of alum sludge was significant (>99%), while the removal capacity of peat was considerably less than 10% after 10 weeks. Both materials significantly reduced the levels of COD, but it was noted that peat had a marginally greater initial P removal capacity (68 ± 22%) and did perform better than alum sludge (57 ± 12%). Moreover, pre-treatment is a crucial step to harness the full potential of peat. Overall, this study provides a scientific clue for sorbents selection when considering alum sludge and peat to maximize their value in practice.

Galectin-3 inhibition suppresses drug resistance, motility, invasion and angiogenic potential in ovarian cancer.

OBJECTIVE: Ovarian cancer is the most deadly gynecologic malignancy worldwide. Since the pathogenesis of ovarian cancer is incompletely understood, and there are no available screening techniques for early detection, most patients are diagnosed with advanced, incurable disease. In an effort to develop innovative and effective therapies for ovarian cancer, we tested the effectiveness of Galecti-3C in vitro. This is a truncated, dominant negative form of Galectin-3, which is thought to act by blocking endogenous Galectin-3. METHODS: We produced a truncated, dominant-negative form of Galectin-3, namely Galetic-3C. Ovarian cancer cell lines and primary cells from ovarian cancer patients were treated with Galectin-3C, and growth, drug sensitivity, and angiogenesis were tested. RESULT: We show, for the first time, that Galectin-3C significantly reduces the growth, motility, invasion, and angiogenic potential of cultured OC cell lines and primary cells established from OC patients. CONCLUSIONS: Our findings indicate that Galectin-3C is a promising new compound for the treatment of ovarian cancer.

Enhancing Neoadjuvant Virotherapy's Effectiveness by Targeting Stroma to Improve Resectability in Pancreatic Cancer.

About one-fourth of patients with pancreatic ductal adenocarcinoma (PDAC) are categorized as borderline resectable (BR) or locally advanced (LA). Chemotherapy and radiation therapy have not yielded the anticipated outcomes in curing patients with BR/LA PDAC. The surgical resection of these tumors presents challenges owing to the unpredictability of the resection margin, involvement of vasculature with the tumor, the likelihood of occult metastasis, a higher ratio of positive lymph nodes, and the relatively larger size of tumor nodules. Oncolytic virotherapy has shown promising activity in preclinical PDAC models. Unfortunately, the desmoplastic stroma within the PDAC tumor microenvironment establishes a barrier, hindering the infiltration of oncolytic viruses and various therapeutic drugs-such as antibodies, adoptive cell therapy agents, and chemotherapeutic agents-in reaching the tumor site. Recently, a growing emphasis has been placed on targeting major acellular components of tumor stroma, such as hyaluronic acid and collagen, to enhance drug penetration. Oncolytic viruses can be engineered to express proteolytic enzymes that cleave hyaluronic acid and collagen into smaller polypeptides, thereby softening the desmoplastic stroma, ultimately leading to increased viral distribution along with increased oncolysis and subsequent tumor size regression. This approach may offer new possibilities to improve the resectability of patients diagnosed with BR and LA PDAC.

Modulation of p38 MAPK signaling enhances dendritic cell activation of human CD4+ Th17 responses to ovarian tumor antigen.

The recent finding that Th17 infiltration of ovarian tumors positively predicts patient outcomes suggests that Th17 responses play a protective role in ovarian tumor immunity. This observation has led to the question of whether Th17 cells could be induced or expanded to therapeutic advantage by tumor vaccination. In this study, we show that treatment of ovarian tumor antigen-loaded, cytokine-matured human dendritic cells (DC) with a combination of IL-15 and a p38 MAP kinase inhibitor offers potent synergy in antagonism of CD4(+) Treg induction and redirection toward CD4(+) Th17 responses that correlate with strong CD8(+) cytotoxic T lymphocyte (CTL) activation. Ovarian tumor antigen-specific CD4(+) T cells secrete high levels of IL-17 and show reduced expression of CTLA-4, PD-1, and Foxp3 following activation with IL-15/p38 inhibitor-treated DC. We further show that modulation of p38 MAPK signaling in DC is associated with reduced expression of B7-H1 (PD-L1), loss of indoleamine 2,3-dioxygenase activity, and increased phosphorylation of ERK 1/2 MAPK. These observations may allow the development of innovative DC vaccination strategies to boost Th17 immunity in ovarian cancer patients.

Th17-inducing dendritic cell vaccines stimulate effective CD4 T cell-dependent antitumor immunity in ovarian cancer that overcomes resistance to immune checkpoint blockade.

BACKGROUND: Ovarian cancer (OC), a highly lethal cancer in women, has a 48% 5-year overall survival rate. Prior studies link the presence of IL-17 and Th17 T cells in the tumor microenvironment to improved survival in OC patients. To determine if Th17-inducing vaccines are therapeutically effective in OC, we created a murine model of Th17-inducing dendritic cell (DC) (Th17-DC) vaccination generated by stimulating IL-15 while blocking p38 MAPK in bone marrow-derived DCs, followed by antigen pulsing. METHODS: ID8 tumor cells were injected intraperitoneally into mice. Mice were treated with Th17-DC or conventional DC (cDC) vaccine alone or with immune checkpoint blockade (ICB). Systemic immunity, tumor associated immunity, tumor size and survival were examined using a variety of experimental strategies. RESULTS: Th17-DC vaccines increased Th17 T cells in the tumor microenvironment, reshaped the myeloid microenvironment, and improved mouse survival compared with cDC vaccines. ICB had limited efficacy in OC, but Th17-inducing DC vaccination sensitized it to anti-PD-1 ICB, resulting in durable progression-free survival by overcoming IL-10-mediated resistance. Th17-DC vaccine efficacy, alone or with ICB, was mediated by CD4 T cells, but not CD8 T cells. CONCLUSIONS: These findings emphasize using biologically relevant immune modifiers, like Th17-DC vaccines, in OC treatment to reshape the tumor microenvironment and enhance clinical responses to ICB therapy.

Identification of AKAP-4 as a new cancer/testis antigen for detection and immunotherapy of prostate cancer.

BACKGROUND: Prostate cancer (PC) is the second most common cancer in older men, after skin cancer. PC is difficult to diagnose because the prostate-specific antigen screening method is associated with many false positives. In addition there is a need to develop new and more effective treatments. Among presently available new treatments, immunotherapy is a promising approach. We investigated the expression of the cancer/testis antigen, AKAP-4, in PC patients to evaluate the possibility of exploiting AKAP-4 as a target for immunotherapy. METHODS: We analyzed normal prostate tissues, 15 patients with PC and the LnCAP PC cell line by immunohistochemistry. We tested AKAP-4 immunogenicity through indirect ELISA on sera from patients and healthy subjects, and we generated in vitro AKAP-4-specific cytotoxic lymphocytes from peripheral blood mononuclear cells. RESULTS: AKAP-4 was shown both at the cytoplasmic and surface levels of the LnCAP PC cell line. AKAP-4 was also highly expressed in PC cells from patients. We detected specific anti-AKAP-4 circulating immunoglobulins in AKAP-4 positive subjects. Using recombinant AKAP-4 loaded autologous dendritic cells, we generated AKAP-4-specific and HLA-I-restricted cytotoxic T lymphocytes able to kill PC cells in vitro. Further characterization indicated a Th-1 skewing in the cytokine secretion profile of these cells. CONCLUSIONS: We demonstrate the aberrant expression of AKAP-4 in PC, which will potentially be developed as a biomarker in PC. We provide evidence that AKAP-4 is a potential target for PC adoptive immunotherapy or anti-tumor vaccination.

CD4+ T-cell response against human papillomavirus type 16 E6 protein is associated with a favorable clinical trend.

The association between the CD8+ T-cell responses to human papillomavirus type 16 (HPV-16) E6 protein and a favorable clinical trend has been demonstrated previously. The roles of human papillomavirus (HPV)-specific CD4+ T-cell responses and of regulatory T-cells (Tregs) were examined. Subjects with a recent history of abnormal Papanicolaou smear were eligible, and colposcopy-guided biopsy was performed at enrollment. Interferon-γ enzyme-linked immunospot assay and fluorescent-activated cell sorter analysis to measure the frequencies of Tregs were performed. Subjects with histological diagnoses of cervical intraepithelial neoplasia 1, 2, or 3 were considered to have short-term persistence of cervical abnormality and were called "persistors" (n = 51) while those of normal histology were designated to be "regressors" (n = 33). A significantly higher percentage CD4+ T-cell response was detected in the regressors (15/33 or 45.5%) compared with the persistors (10/51 or 19.6%) (P = .015) for the E6 peptides but not for the E7 peptides. The CD4+ responses to certain E6 regions [E6(16-40), E6(91-115), E6(106-130), and E6(136-158)] were also significantly higher in the regressors. Although there was no difference in the frequencies of Tregs between the two groups, low frequencies of Tregs were significantly associated with positive CD4+ T-cell responses within certain E6 regions [E6(16-40), E6(31-55), E6(76-100), E6(91-115), and E6(106-130)]. The CD4+ and CD8+ T-cell responses to the HPV-16 E6 protein are associated with a favorable clinical trend. The HPV-16 E6 protein should be incorporated in the design of an HPV therapeutic vaccine.

Repurposing live attenuated trivalent MMR vaccine as cost-effective cancer immunotherapy.

It has long been known that oncolytic viruses wield their therapeutic capability by priming an inflammatory state within the tumor and activating the tumor immune microenvironment, resulting in a multifaceted antitumor immune response. Vaccine-derived viruses, such as measles and mumps, have demonstrated promising potential for treating human cancer in animal models and clinical trials. However, the extensive cost of manufacturing current oncolytic viral products makes them far out of reach for most patients. Here by analyzing the impact of intratumoral (IT) administrations of the trivalent live attenuated measles, mumps, and rubella viruses (MMR) vaccine, we unveil the cellular and molecular basis of MMR-induced anti-cancer activity. Strikingly, we found that IT delivery of low doses of MMR correlates with tumor control and improved survival in murine hepatocellular cancer and colorectal cancer models via increased tumor infiltration of CD8+ granzyme B+ T-cells and decreased macrophages. Moreover, our data indicate that MMR activates key cellular effectors of the host's innate and adaptive antitumor immunity, culminating in an immunologically coordinated cancer cell death. These findings warrant further work on the potential for MMR to be repurposed as safe and cost-effective cancer immunotherapy to impact cancer patients globally.

Th17-inducing autologous dendritic cell vaccination promotes antigen-specific cellular and humoral immunity in ovarian cancer patients.

In ovarian cancer (OC), IL-17-producing T cells (Th17s) predict improved survival, whereas regulatory T cells predict poorer survival. We previously developed a vaccine whereby patient-derived dendritic cells (DCs) are programmed to induce Th17 responses to the OC antigen folate receptor alpha (FRα). Here we report the results of a single-arm open-label phase I clinical trial designed to determine vaccine safety and tolerability (primary outcomes) and recurrence-free survival (secondary outcome). Immunogenicity is also evaluated. Recruitment is complete with a total of 19 Stage IIIC-IV OC patients in first remission after conventional therapy. DCs are generated using our Th17-inducing protocol and are pulsed with HLA class II epitopes from FRα. Mature antigen-loaded DCs are injected intradermally. All patients have completed study-related interventions. No grade 3 or higher adverse events are seen. Vaccination results in the development of Th1, Th17, and antibody responses to FRα in the majority of patients. Th1 and antibody responses are associated with prolonged recurrence-free survival. Antibody-dependent cell-mediated cytotoxic activity against FRα is also associated with prolonged RFS. Of 18 patients evaluable for efficacy, 39% (7/18) remain recurrence-free at the time of data censoring, with a median follow-up of 49.2 months. Thus, vaccination with Th17-inducing FRα-loaded DCs is safe, induces antigen-specific immunity, and is associated with prolonged remission.

Human papillomavirus type 16 and 18 E7-pulsed dendritic cell vaccination of stage IB or IIA cervical cancer patients: a phase I escalating-dose trial.

The safety and immunogenicity of the human papillomavirus type 16 (HPV16) or HPV18 (HPV16/18) E7 antigen-pulsed mature dendritic cell (DC) vaccination were evaluated for patients with stage IB or IIA cervical cancer. Escalating doses of autologous DC (5, 10, and 15 x 10(6) cells for injection) were pulsed with recombinant HPV16/18 E7 antigens and keyhole limpet hemocyanin (KLH; an immunological tracer molecule) and delivered in five subcutaneous injections at 21-day intervals to 10 cervical cancer patients with no evidence of disease after they underwent radical surgery. Safety, toxicity, delayed-type hypersensitivity (DTH) reaction, and induction of serological and cellular immunity against HPV16/18 E7 and KLH were monitored. DC vaccination was well tolerated, and no significant toxicities were recorded. All patients developed CD4(+) T-cell and antibody responses to DC vaccination, as detected by enzyme-linked immunosorbent spot (ELISpot) and enzyme-linked immunosorbent assays (ELISA), respectively, and 8 out of 10 patients demonstrated levels of E7-specific CD8(+) T-cell counts, detected by ELISpot during or immediately after immunization, that were increased compared to prevaccination baseline levels. The vaccine dose did not predict the magnitude of the antibody or T-cell response or the time to detection of HPV16/18 E7-specific immunity. DTH responses to intradermal injections of HPV E7 antigen and KLH were detected for all patients after vaccination. We conclude that HPV E7-loaded DC vaccination is safe and immunogenic for stage IB or IIA cervical cancer patients. Phase II E7-pulsed DC-based vaccination trials with cervical cancer patients harboring a limited tumor burden, or who are at significant risk of tumor recurrence, are warranted.

Generation of CA125-specific cytotoxic T lymphocytes in human leukocyte antigen-A2.1-positive healthy donors and patients with advanced ovarian cancer.

OBJECTIVE: To identify potential immunogenic peptides derived from CA125. STUDY DESIGN: A bioinformatics approach was used to identify peptides derived from CA125 that bind to human leukocyte antigen A2.1 and elicit peptide-specific human cytotoxic T-lymphocyte responses in healthy individuals and patients with ovarian carcinoma. RESULTS: CD8+ cytotoxic T-lymphocyte populations generated against 4 CA125-derived peptides were able to induce lysis of autologous peptide-loaded target cells. CA125 YTLDrDSLYV peptide-specific cytotoxic T lymphocytes were found to effectively kill ovarian tumors expressing CA125. Cytotoxicity was inhibited by antihuman leukocyte antigen A2.1 (BB7-2) and antihuman leukocyte antigen class I (W6/32) antibodies, whereas natural killer-sensitive targets were not lysed. YTLDrDSLYV peptide-specific cytotoxic T lymphocyte precursor frequency was low in peripheral blood leukocytes of normal donors and patients with ovarian cancer as determined by interferon-gamma production in ELISPOT assays. Intracellular cytokine expression measured by flow cytometry showed a type 1 cytokine profile in YTLDrDSLYV peptide-specific cytotoxic T lymphocytes. CONCLUSION: The CA125 YTLDrDSLYV peptide is an immunogenic epitope and may represent an attractive target for immunotherapy of ovarian cancer.

Gastrointestinal Tract Dysbiosis Enhances Distal Tumor Progression through Suppression of Leukocyte Trafficking.

The overall use of antibiotics has increased significantly in recent years. Besides fighting infections, antibiotics also alter the gut microbiota. Commensal bacteria in the gastrointestinal tract are crucial to maintain immune homeostasis, and microbial imbalance or dysbiosis affects disease susceptibility and progression. We hypothesized that antibiotic-induced dysbiosis of the gut microbiota would suppress cytokine profiles in the host, thereby leading to changes in the tumor microenvironment. The induced dysbiosis was characterized by alterations in bacterial abundance, composition, and diversity in our animal models. On the host side, antibiotic-induced dysbiosis caused elongated small intestines and ceca, and B16-F10 melanoma and Lewis lung carcinoma progressed more quickly than in control mice. Mechanistic studies revealed that this progression was mediated by suppressed TNFα levels, both locally and systemically, resulting in reduced expression of tumor endothelial adhesion molecules, particularly intercellular adhesion molecule-1 (ICAM-1) and a subsequent decrease in the number of activated and effector CD8+ T cells in the tumor. However, suppression of ICAM-1 or its binding site, the alpha subunit of lymphocyte function-associated antigen-1, was not seen in the spleen or thymus during dysbiosis. TNFα supplementation in dysbiotic mice was able to increase ICAM-1 expression and leukocyte trafficking into the tumor. Overall, these results demonstrate the importance of commensal bacteria in supporting anticancer immune surveillance, define an important role of tumor endothelial cells within this process, and suggest adverse consequences of antibiotics on cancer control. SIGNIFICANCE: Antibiotic-induced dysbiosis enhances distal tumor progression by altering host cytokine levels, resulting in suppression of tumor endothelial adhesion molecules and activated and effector CD8+ T cells in the tumor.

Protective CD8+ T-cell responses to cytomegalovirus driven by rAAV/GFP/IE1 loading of dendritic cells.

BACKGROUND: Recent studies demonstrate that recombinant adeno-associated virus (rAAV)-based antigen loading of dendritic cells (DCs) generates in vitro, significant and rapid cytotoxic T-lymphocyte (CTL) responses against viral antigens. METHODS: We used the rAAV system to induce specific CTLs against CVM antigens for the development of cytomegalovirus HCMV) gene therapy. As an extension of the versatility of the rAAV system, we incorporated immediate-early 1 (IE1), expressed in HCMV. Our rAAV vector induced a strong stimulation of CTLs directed against the HCMV antigen IE1. We then investigated the efficiency of the CTLs in killing IE1 targeted cells. RESULTS: A significant MHC Class I-restricted, anti-IE1-specific CTL killing was demonstrated against IE1 positive peripheral blood mononuclear cells (PBMC) after one, in vitro, stimulation. CONCLUSION: In summary, single PBMC stimulation with rAAV/IE1 pulsed DCs induces strong antigen specific-CTL generation. CTLs were capable to lyse low doses of peptides pulsed into target cells. These data suggest that AAV-based antigen loading of DCs is highly effective for generating human CTL responses against HCMV antigens.

Endemic Burkitt's lymphoma: a polymicrobial disease?

Endemic Burkitt's lymphoma is the most common childhood cancer in equatorial Africa. Two ubiquitous human pathogens are thought to be responsible for the aetiology of this disease: Epstein-Barr virus and Plasmodium falciparum malaria. New data suggest how these two pathogens might interact to result in disease and provide insights into the emerging concepts of polymicrobial disease pathogenesis.

Regulatory T Cells in Gynecologic Cancer.

Increasing evidence supports that regulatory T cells (Tregs) within the tumor, tumor draining lymph nodes, ascites and peripheral blood of patients with cancer are associated with poor prognosis. Tregs are important mediators of active immune evasion in cancer. In this review, the potential mechanisms of Treg actions and the roles of Tregs specifically in the tumor microenvironment derived from three types of gynecological cancers, cervical, vulvar and ovarian, are described. The correlations between Tregs and clinical immunotherapeutic study outcomes are discussed. Successful modulation of Tregs would likely have significant impact on the effectiveness of immunotherapeutic treatments in cancer patients.

Biotin dependency due to a defect in biotin transport.

We describe a 3-year-old boy with biotin dependency not caused by biotinidase, holocarboxylase synthetase, or nutritional biotin deficiency. We sought to define the mechanism of his biotin dependency. The child became acutely encephalopathic at age 18 months. Urinary organic acids indicated deficiency of several biotin-dependent carboxylases. Symptoms improved rapidly following biotin supplementation. Serum biotinidase activity and Biotinidase gene sequence were normal. Activities of biotin-dependent carboxylases in PBMCs and cultured skin fibroblasts were normal, excluding biotin holocarboxylase synthetase deficiency. Despite extracellular biotin sufficiency, biotin withdrawal caused recurrent abnormal organic aciduria, indicating intracellular biotin deficiency. Biotin uptake rates into fresh PBMCs from the child and into his PBMCs transformed with Epstein Barr virus were about 10% of normal fresh and transformed control cells, respectively. For fresh and transformed PBMCs from his parents, biotin uptake rates were consistent with heterozygosity for an autosomal recessive genetic defect. Increased biotin breakdown was ruled out, as were artifacts of biotin supplementation and generalized defects in membrane permeability for biotin. These results provide evidence for a novel genetic defect in biotin transport. This child is the first known with this defect, which should now be included in the identified causes of biotin dependency.

Treatment of chemotherapy-resistant human ovarian cancer xenografts in C.B-17/SCID mice by intraperitoneal administration of Clostridium perfringens enterotoxin.

Ovarian cancer remains the most lethal gynecologic malignancy in the United States. Although many patients with advanced-stage disease initially respond to standard combinations of surgical and cytotoxic therapy, nearly 90% develop recurrence and inevitably die from the development of chemotherapy-resistant disease. The discovery of novel and effective therapy against chemotherapy-resistant/recurrent ovarian cancer remains a high priority. Using expression profiling, we and others have recently found claudin-3 and claudin-4 genes to be highly expressed in ovarian cancer. Because these tight junction proteins have been described as the low- and high-affinity receptors, respectively, for the cytotoxic Clostridium perfringens enterotoxin (CPE), in this study we investigated the level of expression of claudin-3 and/or claudin-4 in chemotherapy-naive and chemotherapy-resistant primary human ovarian cancers as well as their sensitivity to CPE treatment in vitro. We report that 100% (17 of 17) of the primary ovarian tumors tested overexpress one or both CPE receptors by quantitative reverse transcription-PCR. All ovarian tumors showed a dose-dependent cytotoxic effect to CPE in vitro. Importantly, chemotherapy-resistant/recurrent ovarian tumors were found to express claudin-3 and claudin-4 genes at significantly higher levels when compared with chemotherapy-naive ovarian cancers. All primary ovarian tumors tested, regardless of their resistance to chemotherapeutic agents, died within 24 hours to the exposure to 3.3 microg/mL CPE in vitro. In addition, we have studied the in vivo efficacy of i.p. CPE therapy in SCID mouse xenografts in a highly relevant clinical model of chemotherapy-resistant freshly explanted human ovarian cancer (i.e., OVA-1). Multiple i.p. administration of sublethal doses of CPE every 3 days significantly inhibited tumor growth in 100% of mice harboring 1 week established OVA-1. Repeated i.p. doses of CPE also had a significant inhibitory effect on tumor progression with extended survival of animals harboring large ovarian tumor burdens (i.e., 4-week established OVA-1). Our findings suggest that CPE may have potential as a novel treatment for chemotherapy-resistant/recurrent ovarian cancer.