Search

Aims: To determine whether antigen-independent targeting of the TOP1 inhibitor exatecan to tumor with a pH-sensitive peptide (CBX-12) produces superior synergy with immunotherapy compared with unconjugated exatecan. Materials & methods: In vitro and ex vivo functional assays were performed via FACS and ELISA assays. In vivo efficacy was evaluated in the syngeneic CT26 model. Results: CBX-12 combined with anti-PD-1 or anti-CTLA4 results in delayed tumor growth and complete response, with cured animals displaying long-term antitumor immunity. CBX-12 stimulates expression of MHC 1 and PD-L1 and is an inducer of immunogenic cell death, producing long-term immune recognition of tumor cells and resultant antitumor immunity. Conclusion: The authors' data provide the rationale for exploring immunotherapy combinations with CBX-12 in clinical trials.

Supplementary Figure from TOP1-DNA Trapping by Exatecan and Combination Therapy with ATR Inhibitor

Antisense oligonucleotides are a relatively new therapeutic modality and safety evaluation is still a developing area of research. We have observed that some oligonucleotides can produce acute, nonhybridization dependent, neurobehavioral side effects after intracerebroventricular (ICV) dosing in mice. In this study, we use a combination of

Although combinations of chemotherapy and immunotherapy have shown great promise for cancer treatment, they have also demonstrated significant safety concerns that require dose reductions. Targeting chemotherapy to the tumor can avoid these safety issues, thereby enhancing efficacy of combination therapies. CBX-12 is a novel peptide-drug agent targeting the TOP1-inhibiting drug exatecan to tumor via pH-sensitive peptide. Unlike tumor targeting via antibody, CBX-12 universally targets all solid tumors. CBX-12 avoids the immune cell toxicity of nontumor-targeted exatecan and safely synergizes with immunotherapies. CBX-12 treatment causes tumor cells to express and secrete molecules that result in activation of immune components to recognize and eliminate tumor cells. These data support the upcoming clinical trials of CBX-12 in combination with immunotherapy.

While specific cell signaling pathway inhibitors have yielded great success in oncology, directly triggering cancer cell death is one of the great drug discovery challenges facing biomedical research in the era of precision oncology. Attempts to eradicate cancer cells expressing unique target proteins, such as antibody-drug conjugates (ADCs), T-cell engaging therapies, and radiopharmaceuticals have been successful in the clinic, but they are limited by the number of targets given the inability to target intracellular proteins. More recently, heterobifunctional small molecules such as Proteolysis Targeting Chimera (PROTACs) have paved the way for protein proximity inducing therapeutic modalities.Here, we describe a proof-of-concept study using novel heterobifunctional small molecules calledRegulatedInducedProximityTargetingChimeras or RIPTACs, which elicit a stable ternary complex between a target protein selectively expressed in cancer tissue and a pan-expressed protein essential for cell survival. The resulting cooperative protein:protein interaction (PPI) abrogates the function of the essential protein, thus leading to cell death selectively in cells expressing the target protein. This approach not only opens new target space by leveraging differentially expressed intracellular proteins but also has the advantage of not requiring the target to be a driver of disease. Thus, RIPTACs can address non-target mechanisms of resistance given that cell killing is driven by inactivation of the essential protein.Using the HaloTag7-FKBP model system as a target protein, we describe RIPTACs that incorporate a covalent or non-covalent target ligand connected via a linker to effector ligands such as JQ1 (BRD4), BI2536 (PLK1), or multi-CDK inhibitors such as TMX3013 or dinaciclib. We show that these RIPTACs exhibit positive co-operativity, accumulate selectively in cells expressing HaloTag7-FKBP, form stable target:RIPTAC:effector trimers in cells, and induce an anti-proliferative response in target-expressing cells. We propose that RIPTACs are a novel heterobifunctional therapeutic modality to treat cancers that are known to selectively express a specific intracellular protein.

Exatecan and deruxtecan are antineoplastic camptothecin derivatives in development as tumor-targeted-delivery warheads in various formulations including peptides, liposomes, polyethylene glycol nanoparticles, and antibody–drug conjugates. Here, we report the molecular pharmacology of exatecan compared with the clinically approved topoisomerase I (TOP1) inhibitors and preclinical models for validating biomarkers and the combination of exatecan with ataxia telangiectasia and Rad3-related kinase (ATR) inhibitors. Modeling exatecan binding at the interface of a TOP1 cleavage complex suggests two novel molecular interactions with the flanking DNA base and the TOP1 residue N352, in addition to the three known interactions of camptothecins with the TOP1 residues R364, D533, and N722. Accordingly, exatecan showed much stronger TOP1 trapping, higher DNA damage, and apoptotic cell death than the classical TOP1 inhibitors used clinically. We demonstrate the value of SLFN11 expression and homologous recombination (HR) deficiency (HRD) as predictive biomarkers of response to exatecan. We also show that exatecan kills cancer cells synergistically with the clinical ATR inhibitor ceralasertib (AZD6738). To establish the translational potential of this combination, we tested CBX-12, a clinically developed pH-sensitive peptide–exatecan conjugate that selectively targets cancer cells and is currently in clinical trials. The combination of CBX-12 with ceralasertib significantly suppressed tumor growth in mouse xenografts. Collectively, our results demonstrate the potency of exatecan as a TOP1 inhibitor and its clinical potential in combination with ATR inhibitors, using SLFN11 and HRD as predictive biomarkers.

Historical herbarium collections have been proposed as a last resort for recovery of extinct plant species not represented in dedicated seed banks or other living conservation collections. For critically endangered plants at the brink of extinction, herbarium collections may also contain historical material from extinct subpopulations representing a species' former range and lost genetic diversity of high value for conservation management. We explored the potential for germination of 81 critically endangered seed plant taxa endemic to the Hawaiian island of Kaua'i from herbarium specimens in herbarium PTBG of the National Tropical Botanical Garden (NTBG). Of 1250‐recorded specimens of wild origin, 138 specimens representing 37 taxa contained mature seeds that could be subjected to germination testing. Seven of these taxa were not represented by any NTBG seed bank collections. Fresh embryos were observed in one seed of each of the three species Schiedea helleri, Schiedea kauaiensis, and Viola helena. While potential germination success may be low, we conclude that testing of seeds from herbarium collections should be extended from a focus on strictly extinct taxa to critically endangered taxa, which may not have sufficient representation in seed banks or other living collections of subpopulations and genetic diversity across their wild range.

Infectious bronchitis is a respiratory disease of chickens caused by a gammacoronavirus named infectious bronchitis virus (IBV). In addition to affecting the respiratory tract, IBV may also induce urogenital infections, leading to nephropathogenic disease, false layer syndrome in laying hens, and epididymal lithiasis and epididymitis in males. Here, we report a case of decreased reproductive efficiency due to male infertility in 33- to 38-wk-old broiler breeders. At necropsy, the males presented with urates deposited on the skin around the vent and testicular asymmetry due to marked unilateral atrophy. Histopathology revealed lymphocytic epididymitis, epididymal lithiasis, and orchitis. IBV antigen was detected within collecting and efferent ducts of epididymides by immunohistochemistry. IBV strain DMV/1639 was detected by reverse transcription-quantitative PCR in pools of testes, oviducts, tracheas, cecal tonsils, and kidneys from a 37-wk-old affected flock. This report shows evidence of the role of IBV in male chicken infertility and highlights the importance of performing molecular surveillance of IBV to monitor vaccine strains and to detect emerging variants that can potentially hinder production.

Tau is a microtubule-associated protein (

Topoisomerase inhibitors are potent DNA damaging agents which are widely used in oncology, and they demonstrate robust synergistic tumor cell killing in combination with DNA repair inhibitors, including poly(ADP)-ribose polymerase (PARP) inhibitors. However, their use has been severely limited by the inability to achieve a favorable therapeutic index due to severe systemic toxicities. Antibody-drug conjugates address this issue via antigen-dependent targeting and delivery of their payloads, but this approach requires specific antigens and yet still suffers from off-target toxicities. There is a high unmet need for a more universal tumor targeting technology to broaden the application of cytotoxic payloads. Acidification of the extracellular milieu arises from metabolic adaptions associated with the Warburg effect in cancer. Here we report the development of a pH-sensitive peptide-drug conjugate to deliver the topoisomerase inhibitor, exatecan, selectively to tumors in an antigen-independent manner. Using this approach, we demonstrate potent in vivo cytotoxicity, complete suppression of tumor growth across multiple human tumor models, and synergistic interactions with a PARP inhibitor. These data highlight the identification of a peptide-topoisomerase inhibitor conjugate for cancer therapy that provides a high therapeutic index, and is applicable to all types of human solid tumors in an antigen-independent manner., Graphical Abstract Graphical AbstractCBX-12 is a pH-sensitive peptide-drug conjugate that selectively delivers the TOP1 inhibitor exatecan into tumor cells in an antigen agnostic manner. The peptide forms an alpha helix only in the low pH tumor microenvironment, allowing directional insertion of the peptide across the tumor membrane, cleavage of the linker within the cytosol, and release of free exatecan within the tumor cell. This pH-based delivery approach results in a high therapeutic index universally across solid tumors.

Auristatins such as monomethyl auristatin E (MMAE) are a class of high potency microtubule targeting compounds that have an extremely narrow therapeutic window. Targeting potent auristatins to the tumor is the only feasible method of unlocking the clinical potential of such toxic molecules. While there are currently four marketed antibody-drug conjugates (ADCs) featuring auristatins, these ADCs face the same fundamental issues - tumor restriction by target antigen and the potential for off target release of payload. Alphalex࣪ is a tumor targeting technology consisting of a unique variant of a family of pH-Low Insertion Peptides (pHLIP®) that target acidic cell surfaces (references 1-2), a cleavable small molecule linker, and an anti-cancer agent warhead. Alphalex࣪ thereby allows for antigen independent targeting of the tumor and enables intracellular delivery of the warhead by leveraging the low pH microenvironment of the tumor, a universal feature common to all tumors due to the Warburg effect. Here we report the preclinical efficacy, safety, and antigen-independent tumor-targeting properties of alphalex࣪ conjugated to MMAE. We demonstrate the ability of alphalex࣪-MMAE to display potent in vitro and in vivo efficacy in colorectal, non-small cell lung, and prostate carcinoma cell lines. We further show that alphalex࣪-MMAE efficiently and safely delivers efficacious levels of MMAE selectively to tumor and demonstrates extreme plasma stability, with 0.02% warhead release over 24h in the rat. Based on the excellent preclinical safety and efficacy profile of alphalex࣪-MMAE, Cybrexa will move forward with the goal of initiating IND-enabling studies in 2022. References: 1. Wyatt LC, Lewis JS, Andreev OA, Reshetnyak YK, Engelman DM. Applications of pHLIP Technology for Cancer Imaging and Therapy. Trends Biotechnol. 2017 Jul;35(7):653-664. 2. Wyatt LC, Moshnikova A, Crawford T, Engelman DM, Andreev OA, Reshetnyak YK. Peptides of pHLIP family for targeted intracellular and extracellular delivery of cargo molecules to tumors. Proc Natl Acad Sci USA. 2018 Mar 20;115(12):E2811-E2818. Citation Format: Sophia Gayle, Robert Aiello, Jane Bechtold, Patricia Bourassa, Johanna Csengery, Connor Hagen, Katia Howard, Kelli Jones, Lori Lopresti-Morrow, Robert Maguire, Timothy Paradis, Theresa Pasqualini, Joseph Tweed, Laurie Tylaska, Qing Zhang, Vishwas Paralkar. Development of an alphalex™-auristatin low pH targeting conjugate for the treatment of solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1764.

Immune checkpoint blockade (ICB) in combination with chemotherapy is standard of care for several solid tumors. However, potent chemotherapies such as topoisomerase inhibitors can result in severe dose-limiting toxicities requiring dose reduction, limiting their efficacy. Moreover, leukocytopenia from chemotherapy induces immunosuppression that further limits the full potential of combinations with ICB. The use of targeted chemotherapies such as antibody-drug conjugates (ADCs) demonstrate potential synergy with ICB, however are limited to a defined subset of patients with tumors expressing the target antigen. Cybrexa has reported the development of CBX-12, a peptide-drug conjugate consisting of alphalexTM-exatecan (1). Rather than targeting a specific antigen, alphalexTM consists of a unique variant of pH-Low Insertion Peptide (pHLIP®; 2-4) which targets the low pH environment of the tumor, a universal feature characteristic of all tumors due to the Warburg effect. The alphalexTM component of CBX-12 forms an alpha helix only in low pH conditions, allowing for directional insertion of the peptide within the cancer cell membrane, delivery of C-terminally linked exatecan across the membrane, and subsequent intracellular release of active exatecan via glutathione reduction of the linker, thereby allowing for tumor-specific intracellular delivery in an antigen-independent manner. Here we evaluated the potential for CBX-12 to effectively synergize with PD1 and CTLA4 blockade in multiple syngeneic mouse models without requirement for tumor specific antigens. We found that CBX-12/ICB combination treatment significantly delayed tumor growth, improved survival and led to complete tumor regressions. Mice cured with combined CBX-12/ICB therapy demonstrated formation of long-term immunological memory after in vivo and ex vivo tumor rechallenge. The ability of CBX-12 to induce immunogenic cell death was confirmed by vaccinating syngeneic mice with CBX-12 treated tumor cells and subsequent tumor rechallenge, which demonstrated near total anti-tumor immunity induced by CBX-12. Together, these preclinical data demonstrate the potential for CBX-12 to enhance tumor immunogenicity and potentiate the efficacy of ICB in patients with solid tumors affording a superior, universal tumor targeting mechanism that bypasses the limitations of ADCs. 1. Gayle S et al. 2021.Tumor-selective, antigen-independent delivery of a pH sensitive peptide-topoisomerase inhibitor conjugate suppresses tumor growth without systemic toxicity. NAR Cancer. 2. Rather than targeting a specific antigen, alphalexTM includes a pHLIP® peptide. pHLIP® peptides are a family of pH-Low Insertion Peptides that target acidic cell surfaces. pHLIP® was developed at Yale University and the University of Rhode Island, and is exclusively licensed to pHLIP, Inc. 3. Wyatt LC et al. 2017. Applications of pHLIP Technology for Cancer Imaging and Therapy. Trends Biotech. 4. Wyatt LC et al. 2018. Peptides of pHLIP family for targeted intracellular and extracellular delivery of cargo molecules to tumors. PNAS. Citation Format: Sophia Gayle, Juan Vasquez, Timothy Paradis, Kelli Jones, Ranjini Sundaram, Jinny van Doorn, Viswanathan Muthusamy, Ranjit S. Bindra, Robert J. Aiello, Vishwas Paralkar. CBX-12 (alphalexTM-exatecan) sensitizes tumors to immune checkpoint blockade in an antigen agnostic manner by immune activation [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P258.

Maytansines and their derived maytansinoid DMx compounds are high potency microtubule targeting compounds that have an extremely narrow therapeutic window. Unacceptable dose limiting systemic toxicity has limited the therapeutic potential of these potent anti-oncogenic compounds. Targeting maytansinoids to the tumor is the only feasible method of unlocking the clinical potential of such toxic molecules. To date Trastuzumab-DM1 (Kadcyla®) remains the only approved antibody-maytansinoid conjugate on the market. Most preclinical maytansinoid conjugates to date face the same issues encountered by Kadcyla® - tumor restriction by target antigen and the potential for off target release of payload. alphalexTM is a tumor targeting technology consisting of a unique variant of pH-Low Insertion Peptide (pHLIP®; references 1-3), cleavable small molecule linker and anti-cancer agent warhead. alphalexTM thereby allows for antigen independent targeting of the tumor and enables intracellular delivery of the warhead by leveraging the low pH microenvironment of the tumor, a universal feature common to all tumors due to the Warburg effect. Here we demonstrate the ability to conjugate the maytansinoids DM1 and DM4 to alphalexTM via both direct and linker-mediated conjugation. We have demonstrated the ability of our alphalexTM-DM4 conjugate candidates (CBX-13) to have single digit nanomolar potency in vitro as well as exquisitely potent and long-lasting anti-tumor activity in a HER2-negative xenograft model that is un-targetable by competing therapies. In particular we have demonstrated that CBX-13 safely delivers amounts of maytansinoid in vivo that otherwise result in systemic toxicity and death when dosed as free warhead. Based on the SAR of this first generation of maytansinoid conjugates we are further optimizing our alphalexTM - maytansinoid conjugation strategy with the goal of moving forward with IND-enabling studies in the near future. References 1. Rather than targeting a specific antigen, alphalexTM includes a pHLIP® peptide. pHLIP® peptides are a family of pH-Low Insertion Peptides that target acidic cell surfaces. pHLIP® was developed at Yale University and the University of Rhode Island, and is exclusively licensed to pHLIP, Inc. 2. Wyatt LC, Lewis JS, Andreev OA, Reshetnyak YK, Engelman DM. Applications of pHLIP Technology for Cancer Imaging and Therapy. Trends Biotechnol. 2017 Jul;35(7):653-664. 3. Wyatt LC, Moshnikova A, Crawford T, Engelman DM, Andreev OA, Reshetnyak YK. Peptides of pHLIP family for targeted intracellular and extracellular delivery of cargo molecules to tumors. Proc Natl Acad Sci USA. 2018 Mar 20;115(12):E2811-E2818. Citation Format: Sophia Gayle, Robert Aiello, Jane Bechtold, Patricia Bourassa, Johanna Csengery, Ketaki Deshpande, Kelli Jones, Lori Lopresti-Morrow, Robert Maguire, Dan Marshall, Hunter Moore, Timothy Paradis, Laurie Tylaska, Qing Zhang, Robert Volkmann, Ranjit S. Bindra, Peter M. Glazer, Vishwas Paralkar. Development of alphalex™-toxin low pH targeting conjugates for the treatment of solid tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6242.

Topoisomerase inhibitors are potent DNA damaging agents with great potential as anti-cancer drugs for a wide range of solid tumors. However, dose-limiting toxicities such as myelosuppression and gastric toxicity have prevented them from reaching their full clinical potential. Targeting topoisomerase inhibitors with antibodies (i.e. antibody-drug conjugates; ADCs) may enhance the therapeutic window of these agents, but this approach typically limits applicability to a small subset of patients with tumors expressing the target antigen. We recently developed the alphalexTM tumor-targeting platform to overcome the limitations of ADC-based therapeutic strategies. Rather than targeting a specific antigen, alphalexTM consists of a unique variant of pH-Low Insertion Peptide, (pHLIP®; references 1-3) which targets the low pH environment of the tumor, a universal feature characteristic of all tumors due to the Warburg effect. These alphalexTM conjugates form an alpha helix only in low pH conditions, allowing for insertion of the peptide within the cancer cell membrane, delivery of C-terminal warheads across the membrane, and subsequent intracellular release of the agent via glutathione reduction of the linker, thereby allowing for tumor-specific intracellular delivery in an antigen-independent manner. We report the discovery and development of CBX-12, an alphalexTM conjugate of the potent topoisomerase inhibitor, exatecan. CBX-12 provides additional proof of mechanism to the alphalexTM platform by displaying remarkable tumor-targeting properties in preclinical models. CBX-12 displays enhanced stability in plasma in vivo, undergoing only 0.003% warhead release over 30 hours in circulation and demonstrating exquisite selectivity for tumor over normal tissues in mouse tumor models. Notably, CBX-12 allows for efficient delivery of exatecan into tumors due to a highly optimized cleavable linker, allowing CBX-12 to display extraordinary efficacy in a HER2-negative tumor model in an antigen-independent manner. At 10 mg/kg, CBX-12 treatment almost completely suppressed growth of human colorectal tumors in mice, with complete sparing of bone marrow. In contrast, in animals dosed with the equimolar free exatecan (1.15 mg/kg) there was substantial tumor growth accompanied by neutropenia and weight loss. This superior profile of CBX-12 allow us to greatly enhance efficacy relative to dosing equimolar amounts of unconjugated exatecan, which causes significant, dose-limiting bone marrow toxicity. We have demonstrated that CBX-12 is both safe and has potent anti-tumor activity in preclinical models, and we plan to rapidly move forward with the clinical development of CBX-12 as our lead candidate. References 1. Rather than targeting a specific antigen, alphalexTM includes a pHLIP® peptide. pHLIP® peptides are a family of pH-Low Insertion Peptides that target acidic cell surfaces. pHLIP® was developed at Yale University and the University of Rhode Island and is exclusively licensed to pHLIP, Inc. 2. Wyatt LC et al. Applications of pHLIP Technology for Cancer Imaging and Therapy. Trends Biotechnol. 2017;35(7):653-664. 3. Wyatt LC et al. Peptides of pHLIP family for targeted intracellular and extracellular delivery of cargo molecules to tumors. PNAS. 2018;115(12):E2811-E2818. Citation Format: Robert J. Aiello, Sophia Gayle, Jane Bechtold, Patricia Bourassa, Johanna Csengery, Ketaki Deshpande, Kelli Jones, Lori Lopresti-Morrow, Robert Maguire, Dan Marshall, Hunter Moore, Timothy Paradis, Laurie Tylaska, Qing Zhang, Robert Volkmann, Ranjit S. Bindra, Peter M. Glazer, Vishwas Paralkar. CBX-12: A low pH targeting alphalex™-exatecan conjugate for the treatment of solid tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6249.

Previous studies documented the common occurrence of transitory cyanosis and echocardiographic aortic insufficiency in mature commercial broiler breeder roosters. During further investigations, we observed a high prevalence of hearts exhibiting extensive dilation of the left ventricle chamber compatible with dilated left ventricular cardiomyopathy present in both cyanotic and normal subpopulations. We conducted quantitative studies focused on documentation of cardiac ventricle parameters by using simple gross morphometric methods performed on formalin-fixed hearts obtained from both clinically normal roosters and those exhibiting variable transitory cyanosis, echocardiographic aortic insufficiency, or both. A high prevalence of often dramatic left ventricular dilation reflected in enlarged left ventricular chamber areas and elevated left ventricle-to-total ventricle area ratios was morphometrically documented. However, no statistically significant differences in the occurrence of ventricular abnormalities were observed between normal and cyanotic roosters. Age-associated changes were also demonstrated by comparative morphometric studies on hearts from normal market-age broilers (average age of 7 wk) and those of mature roosters (average age of 42 wk). Elevation in both left and right ventricular weight-to-total heart weight ratios dramatically increased with aging. In addition, values (average ± SD) for the left ventricle chamber area-to-total ventricle area ratios increased from 3.2 ± 2.0% in broilers up to 10.0 ± 8.8% in roosters. None of the normal broilers studied demonstrated left ventricular volume ratios above 10%, whereas 33% of the roosters had left ventricular volume ratios above 10%, including 13% with ratios of 20% or higher. However, the left ventricle wall area-to-body weight ratios were much closer for the two age groups (0.85 ± 0.18 cm(2)/kg in broilers and 0.79 ± 0.13 cm(2)/kg in roosters). Also, the standard right ventricle-to-total ventricle weight ratio (RV/TV) went from 0.18 ± 0.04 in broilers to 0.25 ± 0.12 in roosters, whereas the left ventricle-to-total ventricle weight ratios were similar for the two age groups (0.74 ± 0.12 and 0.75 ± 0.08 in broilers and roosters, respectively). Our results for RV/TV in normal broilers were similar to the reported values for normal market-age broilers. In contrast, 36% of the roosters had RV/TV above values reported for broilers considered reflective of right ventricular hypertrophy due to pulmonary hypertension, whereas 4% had values above the reported threshold for broilers dying with ascites (ratios greater than 0.0249 and 0.299, respectively). However, ascites was not observed for any of the roosters. Although essentially all cardiac morphometric parameters demonstrated statistically significant correlations with the age-class group comparisons, significance could not be documented for comparisons between cardiomorphometrics and the subjective occurrences of transitory cyanosis or echocardiographic aortic insufficiency.

In late spring of 2009 and 2010, there were reports of severe black fly (Simulium spp., shown in Fig. 1) outbreaks in various counties in Mississippi, especially those in and around the Mississippi River Delta. Complaints were of black flies attacking multiple species of backyard poultry and causing high morbidity and mortality in affected flocks. At several affected locations, black flies were readily observed swarming around and feeding on birds. A large number of these parasites were easily trapped on fly strips (Fig. 2). Multifocal to coalescing cutaneous hemorrhagic lesions, consistent with fly bites, were seen on the birds. Upon necropsy examination, a large number of black flies were also observed in the digestive tract (Fig. 3). Although black flies may cause disease directly, such as cardiopulmonary collapse and anaphylactoid reactions, detection of Leucocytozoon in blood smears (Fig. 4) of affected birds prompted further investigations of this protozoan as a cause of disease. Leucocytozoon spp. are known to be transmitted by black flies and may be associated with morbidity and mortality in birds such as poultry. From June 2009 through July 2012, the investigation included a total collection of 1068 individual blood samples, representing 371 individual premises in 89 counties/parishes across Mississippi (59), Alabama (10), Louisiana (4), and Tennessee (16). Of the 371 premises where blood samples were collected, 96 (26%) were either positive or highly suspected to be positive for Leucocytozoon spp. by blood smear analysis, and 5 (1.2%) were positive for Haemoproteus spp. by blood smear analysis. Attempts to diagnose Leucocytozoon spp. by PCR analysis and sequencing were complicated by coinfections with two closely related haemosporidians (Haemoproteus spp. and Plasmodium spp.). A novel technique involving flow cytometry was also explored. This study discusses the black fly field outbreak, the involvement of haemosporidians, molecular methods for detection of both the black flies and blood parasites, and initial attempts at flow cytometry.

In this field experiment, a confederate asked participants to provide a quarter for the confederate to make a telephone call. In the experimental condition, based on the concept of exchange, the confederate offered participants twenty-five pennies for the quarter. The researchers found that there was a significant difference in whether or not the confederate offered the pennies. Eighty-five percent of the participants complied when offered the pennies, while only 35 percent complied without the offer of exchange. Gender was not a factor. Participants over sixty-years-old complied more often than any other age group. Economic status was a factor in that those in the middle range tended to comply more often than those at either the low end or the upper end.

Diabetes mellitus is increasing in frequency and is associated with disabling acute and chronic complications. There is evidence to indicate that excellent glucose control may retard the development and/or progression of these complications. In order to optimize diabetic control, patients are encouraged to monitor their glucose frequently We describe a patient who provided inaccurate glucose monitoring results, delaying effective management of his progressively increasing glycosylated hemoglobin level. The diagnostic clue to his erroneous glucose monitoring results was the lack of intra-day variation in this patient on insulin therapy. Moreover, glucose records within the patient's glucometer pointed to a much less frequent glucose monitoring than the written data provided by the patient. The glucometer was accurate when used by the patient under direct observation. It remains unclear whether this patient deliberately misled his providers or if the erroneous data reflected underlying cognitive dysfunction. Providers are encouraged to approximate average blood sugars based on glycosylated hemoglobin values and compare this to home monitoring results provided by the patient. Primary providers should also expect a certain degree of variability when reviewing home blood sugar values with their patients (on insulin therapy) and consider further investigation should the numbers lack such variation. Clinicians are urged to inspect the actual glucose readings on the patient's glucometer as well as inspecting written glucose records. Observing the patient's technique and accuracy when using their personal glucometer should also be considered.

African Americans have a higher prevalence of Diabetes mellitus and associated complications. The prevalence of Vitamin D deficiency is also higher in African Americans. We report an African American veteran who was followed for a period of 10 years in the Endocrine clinic for insulin-requiring diabetes. Despite intensive, medical, nutritional and educational efforts during that period, no discernible progress was made in achieving any improvement in glycemic control. The patient appeared to be noncompliant with recommended strategies to improve glycemia. The patient was seen recently and was found to be profoundly Vitamin D deficient with a 25 (OH) Vitamin D level of 11.6 ng/ml [30-100 ng/ml]. While patient did not wish to change his insulin regimen or diabetic management, he was willing to accept Vitamin D therapy. Replacement with Vitamin D was associated with significant improvement in glycosylated hemoglobin to previously unmatched levels of glycemic control. We discuss the multiple potential mechanisms by which improved Vitamin D status may result in improved diabetes control. Given the current pandemic of Vitamin D deficiency and the plethora of potential benefits, we recommend maintaining adequate Vitamin D reserves in diabetic patients with a special emphasis on minority populations.