Search

Substance use disorder, and particularly cocaine use disorder, is a complex disease that affects societal, economic, and psychological factors. Endogenous β-endorphin released after prolonged cocaine withdrawal has been reported to activate the accumbal delta-opioid receptor (DOR), leading to attenuated cocaine seeking. However, using DOR β-endorphin activation to treat cocaine use disorder is impractical since β-endorphin does not cross the blood-brain barrier. Also, only activation of the sub-group DOR1 efficiently attenuates craving, as activation of DOR2 yields an opposite effect. Here, we isolated a specific peptide, PEP1, from a phage display peptide library with similar biological properties to β–endorphin, demonstrating specificity for DOR1 and functioning as full receptor agonists. Our pharmacodynamic results showed fast trafficking incorporation of DOR into the cell membrane, interpreted as superior rehabilitation of the receptor and its bioavailability compared to commercial agonists. We administered PEP1, either intrabrain or intranasal, to rats trained to self-administer cocaine. PEP1 induced a significant decrease in cocaine-craving behavior and reinstatement in three different animal models of addiction. Also, PEP1 did not exhibit rewarding properties and did not interfere with the natural reward system. ICP-OES analysis revealed that at least one hour post-administration, PEP1 was retained in the brain rather than in peripheral organs. These findings render PEP1 a potential novel regulator of cocaine craving, especially for being non-addictive. Hence, PEP1 should be further examined as a possible new therapy for substance use disorder.

Cancer therapy, from malignant tumor inhibition to cellular eradication treatment, remains a challenge, especially regarding reduced side effects and low energy consumption during treatment. Hence, phytochemicals as cytotoxic sensitizers or photosensitizers deserve special attention. The dark and photo-response of Yemenite 'Etrog' leaf extracts applied to prostate PC3 cancer cells is reported here. An XTT cell viability assay along with light microscope observations revealed pronounced cytotoxic activity of the extract for long exposure times of 72 h upon concentrations of 175 μg/mL and 87.5 μg/mL, while phototoxic effect was obtained even at low concentration of 10.93 μg/mL and a short introduction period of 1.5 h. For the longest time incubation of 72 h and for the highest extract concentration of 175 μg/mL, relative cell survival decreased by up to 60% (below the IC 50 ). In combined phyto-photodynamic therapy, a reduction of 63% compared to unirradiated controls was obtained. The concentration of extract in cells versus the accumulation time was inversely related to fluorescence emission intensity readings. Extracellular ROS production was also shown. Based on an ATR-FTIR analysis of the powdered leaves and their liquid ethanolic extract, biochemical fingerprints of both polar and non-polar phyto-constituents were identified, thereby suggesting their implementation as phyto-medicine and phyto-photomedicine.

To calibrate a murine model to study premalignant to malignant multiple myeloma, mice were inoculated with different amounts of myeloma cells, and changes in the immune profile were tracked for over 200 days. The model highlights the development of T-cell exhaustion and suppressor before the appearance of clinical symptoms., (© 2023 Wiley-VCH GmbH.)

The rise of biological therapeutics in the global pharmaceuticals market has escalated the demand for quality monoclonal antibodies for healthcare and scientific applications. Reducing costs while enhancing production yields without compromising quality are the main challenges to the growth of this industry today. Over the last two decades non-ionizing radiation has been demonstrated to elicit targeted biological responses in a frequency and dose dependent manner. We hypothesize and design a millimeter wave radiation procedure to enhance the yields of antibody-producing hybridoma cell lines. We demonstrate this method enhances the production of IgA and IgG antibodies from MOPC315.BM and U13.6 cells by a factor of 24.05 ± 3.32 and 1.41 ± 0.03 respectively relative to untreated cells. No treatment associated cytotoxicity was observed in either cell line corroborating physiological viability of irradiated cells. Our results demonstrate proof-of-concept of a novel technique to significantly enhance antibody yields from hybridoma cells which could lead to a reduction in antibody production costs. Further studies will focus on scaling up of this technology and employment of non-contact, tuned electromagnetic stimulation of biological systems for targeted responses., (© 2023 Wiley-VCH GmbH.)

Cancer is a leading cause of mortality today. Sooner a cancer is detected, the more effective is the treatment. Histopathological diagnosis continues to be the gold standard worldwide for cancer diagnosis, but the methods used are invasive, time-consuming, insensitive, and still rely to some degree on the subjective judgment of pathologists. Recent research demonstrated that Attenuated Total Reflection-Fourier Transform Infrared (ATR-FTIR) spectroscopy can be used to determine the metastatic potential of cancer cells by evaluating their membrane hydration. In the current study, we demonstrate that the conversion of ATR-FTIR spectra using multifractal transformation generates a unique number for each cell line's metastatic potential. Applying this technique to murine and human cancer cells revealed a correlation between the metastatic capacity of cancer cells within the same lineage and higher multifractal value. The multifractal spectrum value was found to be independent of the cell concentration used in the assay and unique to the tested lineage. Healthy cells exhibited a smaller multifractal spectrum value than cancer cells. Further, the technique demonstrated the ability to detect cancer progression by being sensitive to the proportional change between healthy and cancerous cells in the sample. This enables precise determination of cancer metastasis and disease progression independent of cell concentration by comparing the measured spectroscopy derived multifractal spectrum value. This quick and simple technique devoid of observer bias can transform cancer diagnosis to a great extent improving public health prognosis worldwide., (© 2023. The Author(s).)

Finding synergistic drug combinations is an important area of cancer research. Here, we sought to rationally design synergistic drug combinations with an inhibitor of BTK kinase, ibrutinib, which is used for the treatment of several types of leukemia. We (a) used a pooled shRNA screen to identify genes that protect cells from the drug, (b) identified protective pathways via bioinformatics analysis of these gene sets, and (c) identified drugs that inhibit these pathways. Based on this analysis, we established that inhibitors of proteasome and mTORC1 could synergize with ibrutinib both in vitro and in vivo. We suggest that FDA-approved inhibitors of these pathways could be effectively combined with ibrutinib for the treatment of chronic lymphocytic leukemia (CLL).

Colorectal cancer (CRC) can been sub-divided, based on the generation of tertiary lymphoid structures (TLS), into CRC with a Crohn's like lymphoid reaction (CLR) representing de novo formation of TLSs or CRC lacking TLSs that show Diffuse Inflammatory infiltration (DII). The association between TLS, early treatment initiation and longer survival highlights the need for deeper patient stratification that could lead to more targeted therapies. We hypothesized that such stratification might be achieved by using digital image analyses. Here we retrospectively analyzed 35 CRC patient samples classified as CLR or DII by digital analysis, focusing on the parameters Fractal dimension, Lacunarity and the textural features Angular second momentum, Correlation, Inverse difference momentum and Entropy. Significant differences in the grades of these parameters between the two patient groups provided preliminary data that additional biophysical information can divide CRC into at least 3 subgroups which encompass CLR and DII. Additional studies are needed to test if this sub-classification aids in the selection of targeted therapy for patients with CRC., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier GmbH. All rights reserved.)

In recent years, mathematical models have developed into an important tool for cancer research, combining quantitative analysis and natural processes. We have focused on Chronic Lymphocytic Leukemia (CLL), since it is one of the most common adult leukemias, which remains incurable. As the first step toward the mathematical prediction of in vivo drug efficacy, we first found that logistic growth best described the proliferation of fluorescently labeled murine A20 leukemic cells injected in immunocompetent Balb/c mice. Then, we tested the cytotoxic efficacy of Ibrutinib (Ibr) and Cytarabine (Cyt) in A20-bearing mice. The results afforded calculation of the killing rate of the A20 cells as a function of therapy. The experimental data were compared with the simulation model to validate the latter's applicability. On the basis of these results, we developed a new ordinary differential equations (ODEs) model and provided its sensitivity and stability analysis. There was excellent accordance between numerical simulations of the model and results from in vivo experiments. We found that simulations of our model could predict that the combination of Cyt and Ibr would lead to approximately 95% killing of A20 cells. In its current format, the model can be used as a tool for mathematical prediction of in vivo drug efficacy, and could form the basis of software for prediction of personalized chemotherapy.

The epidermal growth factor-epidermal growth factor receptor (EGF-EGFR) pathway has become the main focus of selective chemotherapeutic intervention. As a result, two classes of EGFR inhibitors have been clinically approved, namely monoclonal antibodies and small molecule kinase inhibitors. Despite an initial good response rate to these drugs, most patients develop drug resistance. Therefore, new treatment approaches are needed. In this work, we aimed to find a new EGFR-specific, short cyclic peptide, which could be used for targeted drug delivery. Phage display peptide technology and biopanning were applied to three EGFR expressing cells, including cells expressing the EGFRvIII mutation. DNA from the internalized phage was extracted and the peptide inserts were sequenced using next-generation sequencing (NGS). Eleven peptides were selected for further investigation using binding, internalization, and competition assays, and the results were confirmed by confocal microscopy and peptide docking. Among these eleven peptides, seven showed specific and selective binding and internalization into EGFR positive (EGFR+ve) cells, with two of them-P6 and P9-also demonstrating high specificity for non-small cell lung cancer (NSCLC) and glioblastoma cells, respectively. These peptides were chemically conjugated to camptothecin (CPT). The conjugates were more cytotoxic to EGFR+ve cells than free CPT. Our results describe a novel cyclic peptide, which can be used for targeted drug delivery to cells overexpressing the EGFR and EGFRvIII mutation.

Selenium (Se) is incorporated into the body via the selenocysteine (Sec) biosynthesis pathway, which is critical in the synthesis of selenoproteins, such as glutathione peroxidases and thioredoxin reductases. Selenoproteins, which play a key role in several biological processes, including ferroptosis, drug resistance, endoplasmic reticulum stress, and epigenetic processes, are guided by Se uptake. In this review, we critically analyze the molecular mechanisms of Se metabolism and its potential as a therapeutic target for cancer. Sec insertion sequence binding protein 2 (SECISBP2), which is a positive regulator for the expression of selenoproteins, would be a novel prognostic predictor and an alternate target for cancer. We highlight strategies that attempt to develop a novel Se metabolism-based approach to uncover a new metabolic drug target for cancer therapy. Moreover, we expect extensive clinical use of SECISBP2 as a specific biomarker in cancer therapy in the near future. Of note, scientists face additional challenges in conducting successful research, including investigations on anticancer peptides to target SECISBP2 intracellular protein., (© 2021 Wiley Periodicals LLC.)

Current standard frontline therapy for newly diagnosed patients with multiple myeloma (NDMM) involves induction therapy, autologous stem cell transplantation (ASCT), and maintenance therapy. Major efforts are underway to understand the biological and the clinical impacts of each stage of the treatment protocols on overall survival statistics. The most routinely used drugs in the pre-ASCT "induction" regime have different mechanisms of action and are employed either as monotherapies or in various combinations. Aside from their direct effects on cancer cell mortality, these drugs are also known to have varying effects on immune cell functionality. The question remains as to how induction therapy impacts post-ASCT immune reconstitution and anti-tumor immune responses. This review provides an update on the known immune effects of melphalan, dexamethasone, lenalidomide, and bortezomib commonly used in the induction phase of MM therapy. By analyzing the actions of each individual drug on the immune system, we suggest it might be possible to leverage their effects to rationally devise more effective induction regimes. Given the genetic heterogeneity between myeloma patients, it may also be possible to identify subgroups of patients for whom particular induction drug combinations would be more appropriate.

Peptide-drug conjugates (PDCs) are being developed for the targeted delivery of drugs to cancer cells. Several approaches are being followed to enhance their stability in biological solutions. Here we describe an effective method to easily couple PDCs to polyethylene-coated gold nanoparticles. We also outline analytical methods to validate the coupling and assays to measure the stability and cytotoxic efficacy of the conjugates.

Background: Despite chemo-induction therapy and autologous stem cell transplantation (ASCT), the vast majority of patients with Multiple Myeloma (MM) relapse within 7 years and the disease remains incurable. Adoptive Allogeneic T-cell therapy (ATCT) might be curative for MM, however current ATCT protocols often lead to graft versus host disease (GvHD). Transplanting only tumor reactive donor T cells that mediate a graft-versus-myeloma (GvM) but not GvHD may overcome this problem., Methods: We used an MHC-matched/miHA-disparate B10.D2 → Balb/c bone marrow transplantation (BMT) murine model and MOPC315.BM MM cells to develop an ATCT protocol consisting of total body irradiation, autologous-BMT and infusion of selective, myeloma-reactive lymphocytes of T cell receptor (TCR) Vβ 2, 3 and 8.3 families (MM-auto BMT ATCT)., Results: Pre-stimulation ex vivo of allogeneic T cells by exposure to MOPC315.BM MM cells in the presence of IL-2, anti-CD3 and anti-CD28 resulted in expansion of the myeloma-reactive T cell TCRVβ 2, 3 and 8.3 subfamilies. Their isolation and infusion into MM-bearing mice resulted in a vigorous GvM response without induction GvHD and long-term survival. Repeated infusion of naïve myeloma-reactive T cell TCRVβ 2, 3 and 8.3 subfamilies was also effective., Conclusions: These data demonstrate that a transplantation protocol involving only selective tumor-reactive donor T cell families is an effective immunotherapy and results in long-term survival in a mouse model of human MM. The results highlight the need to develop similar ATCT strategies for MM patients that result in enhanced survival without symptoms of GvHD.

Pancreatic cancer poorly responds to available drugs, and finding novel approaches to target this cancer type is of high significance. Here, based on a common property of pancreatic cancer cells to express somatostatin receptors (SSTR), we designed drug conjugates with novel somatostatin-derived cyclic peptides (SSTp) with broad selectivity towards SSTR types to facilitate drug targeting of the pancreatic cancer cells specifically. Uptake of our newly designed SSTps was facilitated by SSTRs expressed in the pancreatic cancers, including SSTR2, SSTR3, SSTR4 and SSTR5. Three major drugs were conjugated to our best SSTps that served as delivery vehicles, including Camptothecin (CPT), Combretastatin-4A (COMB) and Azatoxin (AZA). All designed drug conjugates demonstrated penetration to pancreatic cancer cell lines, and significant toxicity towards them. Furthermore, the drug conjugates specifically accumulated in tumors in the animal xenograft model, though some accumulation was also seen in kidney. Overall these findings lay the basis for development of novel drug series that could target the fatal pancreatic cancer., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Background: Peptide-drug-conjugates (PDCs) are being developed as an effective strategy to specifically deliver cytotoxic drugs to cancer cells. However one of the challenges to their successful application is the relatively low stability of peptides in the blood, liver and kidneys. Since AuNPs seem to have a longer plasma half-life than PDCs, one approach to overcoming this problem would be to conjugate the PDCs to gold nanoparticles (AuNPs), as these have demonstrated favorable physico-chemical and safety properties for drug delivery systems. We set out to test whether PEG coated-AuNPs could provide a suitable platform for the non-covalent loading of pre-formed PDCs and whether this modification would affect the bioavailability of the PDCs and their cytotoxicity toward target cancer cells., Methods: Peptides specifically internalized by A20 murine lymphoma cells were isolated from a phage library displaying 7mer linear peptides. Peptide specificity was validated by flow cytometry and confocal microscopy. PDCs were synthesized containing a selected peptide (P4) and either chlorambucil (Chlor), melphalan (Melph) or bendamustine (Bend). Gold nanoparticles were sequentially coated with citrate, PEG-6000 and then PDC (PDC-PEG-AuNP). The physico-chemical properties of the coated particles were analyzed by electrophoresis, TEM, UV-VIS and FTIR. Stability of free and PDC-coated AuNP was determined., Results: Biopanning of the phage library resulted in discovery of several novel peptides that internalized into A20 cells. One of these (P4) was used to synthesize PDCs containing either Chlor, Melph or Bend. All three PDCs specifically killed A20 target cells, however they had short half-lives ranging from 10.6 to 15.4 min. When coated to PEG-AuNPs, the half-lives were extended to 21.0-22.3 h. The PDC-PEG-AuNPs retained cytotoxicity towards the target cells. Moreover, whereas pre-incubation for 24 h of free PDCs almost completely abolished their cytotoxic activity, the PDC-PEG-AuNPs were still active even after 72 h pre-incubation., Conclusions: Peptide-drug-conjugates hold potential for improving the target efficacy of chemotherapeutic drugs, however their short half-lives may limit their application. This hurdle can be overcome by easily conjugating them to gold nanoparticles. This conjugation also opens up the possibility of developing slow release formulations of targeted drug delivery systems containing PDCs.

Metastatic castration-resistant prostate cancer (mCRPC) remains essentially incurable. Targeted Drug Delivery (TDD) systems may overcome the limitations of current mCRPC therapies. We describe the use of strict criteria to isolate novel prostate cancer cell targeting peptides that specifically deliver drugs into target cells. Phage from a libraries displaying 7mer peptides were exposed to PC-3 cells and only internalized phage were recovered. The ability of these phage to internalize into other prostate cancer cells (LNCaP, DU-145) was validated. The displayed peptides of selected phage clones were synthesized and their specificity for target cells was validated in vitro and in vivo. One peptide (P12) which specifically targeted PC-3 tumors in vivo was incorporated into mono-drug (Chlorambucil, Combretastatin or Camptothecin) and dual-drug (Chlorambucil/Combretastatin or Chlorambucil/Camptothecin) PDCs and the cytotoxic efficacy of these conjugates for target cells was tested. Conjugation of P12 into dual-drug PDCs allowed discovery of new drug combinations with synergistic effects. The use of strict selection criteria can lead to discovery of novel peptides for use as drug carriers for TDD. PDCs represent an effective alternative to current modes of free drug chemotherapy for prostate cancer., (Copyright © 2017. Published by Elsevier B.V.)

The resistance of cancer cells to chemotherapeutic agents, whether through intrinsic mechanisms or developed resistance, motivates the search for new chemotherapeutic strategies. In the present report, we demonstrate a facile synthetic strategy towards the discovery of new anti-cancer substances. This strategy is based on simple covalent coupling between known anti-cancer drugs, which results in novel 'chimeric' small molecules. One of these novel compounds, CM358, is the product of an amide bond formation between the known Topoisomerase II (Topo II) inhibitor amonafide (AM) and the known DNA mustard alkylator chlorambucil (CLB). It demonstrates significant enhanced cytotoxicity over an equimolar mixture of AM and CLB in various cancer cell lines and in a xenograft model of human metastatic melanoma. Topo II inhibition as well as in silico docking studies suggest that CM358 is a stronger Topo II binder than AM. This may be attributed, at least partially, to the placement of the CLB moiety in a favorable orientation with respect to DNA cross-linking with nearby guanines. In a human metastatic melanoma (WM 266-4) xenograft model, this compound was profoundly superior to a mixture of AM and CLB in reduction of tumor growth, maintenance of body weight and extension of overall survival., (Copyright © 2017 Elsevier Masson SAS. All rights reserved.)

Cerebrospinal fluid (CSF) is a clear and colorless biological fluid which circulates within brain ventricles (cavities), the spinal cord's central canal, the space between the brain and the spinal cord, as well as their protective coverings, the meninges. Cerebrospinal fluid contains different constituents, such as albumin and lactate, whose levels are used clinically as biomarkers of neurodegenerative disorders. In current clinical practice, analysis of CSF content for the diagnosis of central nervous system disorders requires an invasive procedure known as lumbar puncture or spinal tap. With the aim of developing a noninvasive alternative, we report here the spectral behavior of albumin and lactate over a broad wavelength range of 600-2000 nm, after each was added separately at varying normal and abnormal concentration levels to artificial CSF ( aCSF). Spectral measurements were conducted simultaneously by two different spectrometers working at different spectral ranges in transmittance mode. Spectral analysis revealed that albumin and lactate each possesses its own first and second derivative absorbance spectra fingerprint between 1660 and 1810 nm. Distinguishing albumin from lactate by their spectral data enabled the differentiation between aCSF conditions modeling different neurological disorders. Spectral changes of each compound strongly correlated ( R 2  > 0.9) with absorbance derivative spectra peaks at specific wavelengths, when analyzed by linear regression with variations in their concentration. These findings suggest the feasibility of CSF biomarker assessment by broadband infrared spectroscopy.

To enhance the efficacy of targeted drug delivery, four new peptide-ligand conjugates were synthesized, each consisting of a cyclic RGDfK penta-peptide loaded with two anticancer drugs. The drug release profiles in different media of these new compounds and their cytotoxic activity against melanoma and non-small lung cancer cell lines were evaluated and compared with those of their singly loaded analogs. The cyclic RGDfK penta-peptide was selected as a targeting moiety because of its high affinity and selectivity to the α v β 3 integrin receptor, which is frequently over-expressed in various types of cancer cells. The peptide's core was modified at the side chain of its Lys residue by coupling it with a sixth amino acid (AA) - either Lys (5a) or Ser (5b) (Lys/Ser splitter), resulting in two functional sites which enabled the loading of two therapeutic equivalents onto a single targeting carrier. Using Lys as a splitter resulted in two primary amines. Consequently, conjugates 1a and 1b were synthesized by coupling of 2 Chlorambucils (CLBs) or 2 Camptothecins (CPTs), respectively, to the primary amines of 5a. Conjugate 1c was synthesized from 5b by loading two equivalents of CLB on the amine and the hydroxyl of the Ser splitter, resulting in a homodimeric system with two distinct conjugation sites - amide and ester. The heterodimeric conjugate 1d of CLB and CPT was synthesized by loading each one of the primary amines of 5a with two different drugs - CLB and CPT. The doubling of drug equivalents loaded onto the targeting peptide correlated with enhanced cytotoxic efficacy of the conjugates towards cancer cells. The versatility of chemical linkages of the drugs to the peptides resulted in conjugates with different drug release profiles. Molecular dynamics simulations performed on conjugate 1d demonstrated that this compound occupies a conformational space similar to the bio-active conformation of an integrin-bound cyclic RGD peptide reference peptide (c(RGDf(NMe)V). The modified position in 1d (relative to the reference peptide) points away from the integrin, leading us to hypothesize that this peptide binds the integrin in a manner similar to that of the reference peptide thereby fulfilling a crucial requirement for targeted delivery. The strategy of dual drug loading on a single peptide carrier, gives rise to drugs with different mechanisms of action and release profiles, thus substantially increasing the efficacy of selective killing of tumor cells and while reducing the risk of the development of drug resistance. © 2015 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 160-171, 2016., (© 2015 Wiley Periodicals, Inc.)

New cyclic RGD peptide-anticancer agent conjugates, with different chemical functionalities attached to the parent peptide were synthesized in order to evaluate their biological activities and to provide a comparative study of their drug release profiles. The Integrin binding c(RGDfK) penta-peptide was used for the synthesis of Camptothecin (CPT) carbamate and Chlorambucil (CLB) amide conjugates. Substitution of the amino acid Lys with Ser resulted in a modified c(RGDfS) with a new attachment site, which enabled the synthesis of an ester CLB conjugate. Functional versatility of the conjugates was reflected in the variability of their drug release profiles, while the conserved RGD sequence of a selective binding to the αv integrin family, likely preserved their recognition by the Integrin and consequently their favorable toxicity towards targeted cancer cells. This hypothesis was supported by a computational analysis suggesting that all conjugates occupy conformational spaces similar to that of the Integrin bound bio-active parent peptide., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Bi-nuclear amino acid platforms loaded with various drugs for conjugation to a peptide carrier were synthesized using simple and convenient orthogonally protective solid-phase organic synthesis (SPOS). Each arm of the platform carries a different anticancer agent linked through the same or different functional group, providing discrete chemo- and bio-release profiles for each drug, and also enabling "switch off/switch on" regulation of drug cytotoxicity by conjugation to the platform and to a cell targeting peptide. The versatility of this approach enables efficient production of drug-loaded platforms and determination of favorable drug combinations/modes of linkage for subsequent conjugation to a carrier moiety for targeted cancer cell therapy. The results presented here potentiate the application of amino acid platforms for targeted drug delivery (TDD)., (Copyright © 2014 Elsevier Masson SAS. All rights reserved.)

Targeting drugs through small-molecule carriers with a high affinity to receptors on cancer cells can overcome the lack of target cell specificity of most anticancer drugs. These targeted carrier-drug conjugates are also capable of reversing drug resistance in cancer cells. Although many targeted drug delivery approaches are being tested, the linkage of several and different drugs to a single carrier molecule might further enhance their therapeutic efficacy, particularly if the drugs are engineered for variable time release. This report shows that murine B-cell leukemic cells previously resistant to a chemotherapeutic drug can be made sensitive to that drug as long as it is conjugated to a targeting peptide and, in particular, when the conjugate contains multiple copies of the drug. Using a 13mer peptide (VHFFKNIVTPRTP) derived from the myelin basic protein (p-MBP), dendrimer-based peptide conjugates containing one, two, or four molecules of chlorambucil were synthesized. Although murine hybridomas expressing antibodies to either p-MBP (MBP cells) or a nonrelevant antigen (BCL-1 cells) were both resistant to free chlorambucil, exposure of the cells to the p-MBP-chlorambucil conjugate completely reversed the drug resistance in MBP, but not BCL-1 cells or normal spleen cells. Moreover, at equivalent drug doses, there was significant enhancement in the cytotoxic activity of multidrug versus single-drug copy conjugates. On the basis of these results, the use of multifunctional dendrone linkers bearing several covalently bound cytotoxic agents allows the development of more effective targeted drug systems and enhances the efficacy of currently approved drugs for B-cell leukemia.

Therapeutic monoclonal antibody (TMA) based therapies for cancer have advanced significantly over the past two decades both in their molecular sophistication and clinical efficacy. Initial development efforts focused mainly on humanizing the antibody protein to overcome problems of immunogenicity and on expanding of the target antigen repertoire. In parallel to naked TMAs, antibody-drug conjugates (ADCs) have been developed for targeted delivery of potent anti-cancer drugs with the aim of bypassing the morbidity common to conventional chemotherapy. This paper first presents a review of TMAs and ADCs approved for clinical use by the FDA and those in development, focusing on hematological malignancies. Despite advances in these areas, both TMAs and ADCs still carry limitations and we highlight the more important ones including cancer cell specificity, conjugation chemistry, tumor penetration, product heterogeneity and manufacturing issues. In view of the recognized importance of targeted drug delivery strategies for cancer therapy, we discuss the advantages of alternative drug carriers and where these should be applied, focusing on peptide-drug conjugates (PDCs), particularly those discovered through combinatorial peptide libraries. By defining the advantages and disadvantages of naked TMAs, ADCs and PDCs it should be possible to develop a more rational approach to the application of targeted drug delivery strategies in different situations and ultimately, to a broader basket of more effective therapies for cancer patients.

The increasing resistance of bacteria to antibiotics is a serious problem, caused in part by excessive and improper use of these drugs. One alternative to traditional antibiotic therapy is photodynamic antimicrobial chemotherapy (PACT) which is based on the use of a photosensitizer (PS), activated by illumination with visible light. The poor penetration of visible light through the skin limits the application of PACT to the treatment of skin infections or the use of invasive procedures. To overcome this problem we report the exploitation of light emitted as a result of the chemiluminescent reaction of luminol to excite the PS and we call this process chemiluminescent photodynamic antimicrobial therapy (CPAT). We studied the effect of free and liposome-encapsulated PS (methylene blue or toluidine blue) on bacteria under excitation by either white external light or chemiluminescence emitted by free or liposome-enclosed luminol. PACT showed slightly better performance that CPAT for free and encapsulated PS for both types of bacteria. CPAT resulted in a three log suppression of Staphylococcus aureus and two log suppression of Escherichia coli growth. The use of CPAT may prove to be a novel and more effective form of antimicrobial therapy, particularly for internal infections not easily accessible to traditional PACT., (© 2010 The Authors. Journal Compilation. The American Society of Photobiology.)

Equipment that generates microwave radiation (MWR) spanning the frequency range of 300 MHz-100 GHz is becoming more common. While MWR lacks sufficient energy to break chemical bonds, the disagreement as to whether MWR exposure is detrimental to cellular dysfunction may be difficult to clarify using complex systems such as whole animals, cells, or cell extracts. Recently, the high frequency range of terahertz (THz) radiation has been explored and sources of radiation and its detectors have been developed. THz radiation is associated with the frequency interval from 100 GHz to 20 THz and constitutes the next frontier in imaging science and technology. In the present study, we investigated the effect of radiation in the low frequency THz range (100 GHz) on two defined molecular interactions. First, the interaction of soluble or immobilized calf alkaline phosphatase with the substrate p-nitrophenylphosphate and second, the interaction between an antibody (mouse monoclonal anti-DNP) and its antigen (DNP). Irradiation of enzyme either prior to addition of substrate or during the enzymatic reaction resulted in small but significant reductions in enzyme activity. These differences were not observed if the enzyme had previously been immobilized onto plastic microwells. Exposure of immobilized antigen to radiation did not influence the ability of the antigen to interact with antibody. However, irradiation appeared to decrease the stability of previously formed antigen-antibody complexes. Our data suggest that 100 GHz radiation can induce small but statistically significant alterations in the characteristics of these two types of biomolecular interactions., ((c) 2008 Wiley-Liss, Inc.)

Multiple myeloma is currently an incurable cancer of plasma B cells often characterized by overproduction of abnormally high quantities of a patient-specific, clonotypic immunoglobulin "M-protein." The M-protein is expressed on the cell membrane and secreted into the blood. We previously showed that ligand-toxin conjugates (LTC) incorporating the ribosome-inactivating Ricin-A toxin were very effective in specific cytolysis of the anti-ligand antibody-bearing target cells used as models for multiple myeloma. Here, we report on the incorporation of the membrane-disruptive Cyt1Aa toxin from Bacillus thuringiensis subsp. israelensis into LTCs targeted to murine myeloma cells. Proteolytically activated Cyt1Aa was conjugated chemically or genetically through either its amino or carboxyl termini to the major peptidic epitope VHFFKNIVTPRTP (p87-99) of the myelin basic protein. The recombinant fusion-encoding genes were cloned and expressed in acrystalliferous B. thuringiensis subsp. israelensis through the shuttle vector pHT315. Both chemically conjugated and genetically fused LTCs were toxic to anti-myelin basic protein-expressing murine hybridoma cells, but the recombinant conjugates were more active. LTCs comprising the Cyt1Aa toxin might be useful anticancer agents. As a membrane-acting toxin, Cyt1Aa is not likely to induce development of resistant cell lines.

Photodynamic therapy (PDT) involves a two-stage process. A light-absorbing photosensitiser (Ps) is endocytosed and then stimulated by light, inducing transfer of energy to a cytoplasmic acceptor molecule and the generation of reactive oxygen species that initiate damage to cellular membrane components and cytolysis. The expanded use of PDT in the clinic is hindered by the lack of Ps target-cell specificity and the limited tissue penetration by external light radiation. This study demonstrates that bioconjugates composed of transferrin and haematoporphyrin (Tf-Hp), significantly improve the specificity and efficiency of PDT for erythroleukemic cells by a factor of almost seven-fold. Fluorescence microscopy showed that the conjugates accumulate in intracellular vesicles whereas free Hp was mostly membrane bound. Experiments with cells deliberately exposed to Tf-Hp at

The paper describes a hierarchical design approach to an immunoassay. A morphology for an immunoassay technology is considered as a basis to generate system versions. A 5-stage technology is analyzed. The problem is: Find the best composite version for each stage while taking into account requirements (criteria) at each stage and compatibility between selected design alternatives at different stages. Hierarchical solving procedure consists of two parts: (a) multicriteria ranking of alternative versions at each stage (e.g., selection of Pareto-effective local decisions), (b) composition of the selected versions into a parallel-series composite system (technology). A realistic numerical example illustrates the approach.

Many types of leukemia including multiple myeloma remain essentially incurable despite recent developments in immuno- and chemotherapy. The effectiveness of these therapies might be greatly enhanced by targeting cell surface proteins unique to the malignant clone, which for leukemias of the B cell lineage means clonotypic surface immunoglobulin (sIg). As this immunoglobulin (Ig) is necessarily epitope specific, we are developing ligand-toxin conjugates (LTCs) as a strategy for delivering toxins and other drugs to clonotypic tumor cells. Here we report in vitro studies that illustrate the effectiveness of this approach. LTC comprising the DNP hapten conjugated to ricin A toxin (DNP-RTA) were shown to specifically and effectively kill anti-DNP secreting murine hybridoma (U7.6) cells but not other hybridoma cells (1B12), a murine erythroleukemia cell line (Friend's Leukemia or) normal mouse spleen cells. In addition to direct toxicity, LTC treatment negatively affected the growth characteristics of the few surviving cells as reflected in decreased growth index and an increase in growth inhibition over 72 h post treatment. Interestingly, U7.6 cells that survived one or two LD90 dose(s) of LTC showed no alteration in their dose response to a subsequent attack of LTC indicating that this treatment strategy may not induce drug resistance. These data suggest that LTC therapy may be a new and effective strategy for specific destruction of tumor cells such as myeloma plasma cells and could be extended to other tumors where clonotypic receptors can be identified.

Many elution buffers are in use for the retrieval of proteins from affinity columns. While the aim of these buffers is to dissociate the various chemical bonds that make up protein-protein interactions and return the target protein to the mobile phase in active form, there is considerable difference of opinion as to which buffer is more suitable for particular applications. This review examines the chemical effect of various elution buffers on protein-protein interactions in the context of affinity chromatography and examines strategies that may be used for selection of an appropriate buffer.

Antibodies (Ab) are commonly used in affinity chromatography (AC) as a versatile and specific means of isolating target molecules from complex mixtures. A number of procedures have been developed to immobilize antibodies on the solid matrix. Some of these methods couple the antibody via chemical groups that may be important for specific recognition of antigen, resulting in loss of functionality in a proportion of the antibodies. In other methods, the outcome of immobilization is coupling via unique sites in the Fc region of the antibody molecule, ensuring orientation of the antibody combining sites (Fab) towards the mobile phase. This review discusses the advantages and disadvantages of the various methods available for immobilization and outlines protocols for site-directed, covalent coupling of the antibody to the solid phase that essentially retains the activity of the antibody.

The immobilization of antibodies onto solid phases in an efficient and activity-retaining form is an important goal for both research and industry. Methods have been developed for the site-directed attachment of antibodies to agarose by oxidation of the carbohydrate moieties in their Fc region. Similar attachment to silianized supports have not been as successful. Here we describe a novel combination protocol for the site-directed attachment of periodate oxidized, goat polyclonal antibodies to glass wool fibers activated with 3-aminopropyltriethoxysilane. The study demonstrates that this procedure results in effective immobilization of polyclonal antibodies that retain their antigen-binding capacity. This protocol should prove useful in the development of more efficient and effective glass-based immunosupports.

Only a limited number of erythroid cell surface markers have been described in the literature. Ag-Eb was originally described as an erythroid-specific cell surface glycoprotein and could be used as an erythroid differentiation marker, but more recent studies suggest this localization is more widespread. From the data summarized in this review, it is hypothesized that Ag-Eb is a member of a subset of the transferrin receptor family and that it functions together with these receptors in the uptake and metabolism of iron, particularly at histo-hematic barriers.

An interspecific marker of mammalian erythroid cells, which was called the erythroblast antigen, was identified in 1974, using polyclonal monospecific antibodies. Further studies have demonstrated the expression of this antigen in a variety of nonhemopoietic organs and tissues, which have the following common feature: they have a barrier location; that is, they are located at the boundary. It has been proposed that the erythroblast antigen participates directly or indirectly in the transport of various substances and specifically transport of iron. The present review deals with this topic.

In an attempt to define the autoimmune status of members of multicase families with schizophrenia, sera of both patients and healthy relatives from 28 such cases were tested for antinuclear antibodies, anti-double-stranded DNA, and anti-single-stranded DNA autoantibodies. These autoantibodies were significantly more frequent in both schizophrenic patients and healthy relatives than in normal subjects. Immunoglobulin (Ig) M anti-DNA antibodies were more common in patients, whereas in healthy relatives, IgG anti-DNA antibodies were more common. No significant differences were found between schizophrenic patients and their healthy relatives. The data indicate that an autoimmune process may be involved in the etiology of a subset of patients with schizophrenia.

In this investigation 98 children (median age 24 months) with cows' milk allergy (CMA) were studied over a median period of 2 years to see whether acquisition of clinical tolerance to cows' milk was associated with the changes in levels of IgG and IgE anti-cows' milk antibodies, and skin test reactivity to a cows' milk extract. Two groups of CMA patients were examined. The first were IgE sensitized and responded rapidly to small volumes of cows' milk with urticaria, and/or exacerbations of eczema, and/or wheeze, and/or vomiting (n = 69). The second, a late reacting group (n = 29) demonstrated coughing, diarrhoea, eczematoid rashes, and/or a combination of these which developed more than 20 hr after commencing normal volumes of cows' milk. Significant immunological changes were confined to the 69 IgE sensitized immediate-reacting-group of patients. Of these, there were 15 children who achieved clinical tolerance to cows' milk and they showed a significant fall in the levels of skin test reactivity to cows' milk over the study period (P < 0.01). In addition, these 15 children had lower serum IgE antibodies to cows' milk proteins both at the outset and the final follow-up compared with the 54 patients whose CMA persisted. No consistent change in the IgG antibody responses to cows' milk proteins was seen in either group of patients over the study period. The findings suggest patients with immediate type hypersensitivity to cows' milk proteins whose disease persists for more than 2 years have a more severe dysregulation of IgE synthesis to cows' milk proteins from the outset.(ABSTRACT TRUNCATED AT 250 WORDS)

Many laboratories have established ELISAs for the the routine detection of anti-cardiolipin antibodies (ACA). Earlier studies had indicated that assay incubation at 37 degrees C may interfere with the antigen binding capacity of these antibodies. We have reexamined this phenomenon by comparing ACA titers obtained when incubations are performed at either 37 degrees C or at room temperature (RT). In addition, the effect of coating antigen in aqueous or organic solution was compared. The sera tested included a set of recognized ACA standards and samples from 19 patients with SLE, two with primary anti-phospholipid syndrome, 71 patients with a variety of autoimmune and non-autoimmune disorders and 210 blood bank controls. The results show that while some sera do perform better under either incubation temperature there was no correlation between ACA titers and incubation temperature on a population basis either for IgG or IgM isotypes. This was seen both for positive standards and patient sera. For IgG ACA a similar phenomenon was seen if the microplates were coated with cardiolipin either in sodium carbonate or ethanol. For IgM ACA there was a significant increase in ACA titers at RT when cardiolipin was coated in ethanol. The data suggest that for most sera neither the antigen coating medium nor the assay incubation temperature are important variables in the determination of IgG ACA. Factors contributing to the influence of either variable in individual sera could not be identified.

This paper describes the development of a simple, sensitive and specific enzyme-linked immunosorbent assay (ELISA) for the measurement of serum IgG, IgA and IgM antibodies to cow's milk protein. The specificity of this assay was determined by preparing immunosorbents of cow's milk and human serum albumin (HSA). Fractionation of the antiserum-enzyme label by gel filtration and affinity chromatography showed that the conjugate preparation contained moieties of antiserum-enzyme, and antiserum-enzyme polymers. The preparation also contained enzyme-enzyme and antiserum-antiserum conjugates. Purification of the labeled reagent did not improve the sensitivity. The reagents and conditions used have been analyzed for purposes of standardisation.

We assessed the relationships of clinical symptoms and serum antibody levels during follow-up of 47 patients, aged 3 to 66 months, who were shown by formal milk challenge to have cow milk allergy. Three groups of patients were identified. Group 1 patients (n = 15) were sensitized to IgE and responded rapidly to small volumes of milk with urticaria, an exacerbation of eczema, wheeze, or vomiting. In the second group (n = 24), symptoms of milk enteropathy (vomiting and diarrhea) developed between 1 and 20 hours after milk ingestion. In the group 3 patients (n = 8), coughing, diarrhea, eczematoid rashes, or a combination of these developed more than 20 hours after normal volumes of milk were given. Serum levels of IgG, IgA, IgM, and IgE and of milk-specific anti-cow milk antibodies of these isotypes were measured initially and then at a median follow-up time of 16 months (range 6 to 39 months). In this investigation, changes in these immunologic measures during the study period were related to whether or not clinical tolerance to cow milk was achieved. At follow-up, six patients from group 1, ten from group 2, and two from group 3 were milk tolerant. No consistent change in any of the immunologic measurements was associated with remission of the disease. These findings raise the question of whether acquisition of clinical tolerance to cow milk in cow milk allergy can be attributed solely to immunologic events.

In 47 infants and children aged 4-66 months with clinically proven cow's milk allergy and in a group of age-matched controls, serum IgG, IgA and IgM cow's milk-specific antibodies were determined with ELISA assays while IgE cow's milk-specific antibodies were measured with Pharmacia RAST. The patients were divided into three separate groups according to the time of clinical response to a standardized cow's milk challenge protocol. Immediate reactions (less than 45 min after challenge), which were mainly accompanied by urticarial skin eruptions, were associated with elevated IgE milk-specific antibody levels, indicating the involvement of an immediate hypersensitivity mechanism. Alternatively, intermediate reactions (1-20 h after challenge), which were mainly accompanied by vomiting and diarrhea, were not IgE-mediated. In the late reactions (greater than 20 h after challenge) both eczematous and gastrointestinal reactions were seen. Patients with eczematous eruptions also showed elevated IgE milk-specific antibody levels. IgG milk-specific antibody levels were similar in each of the patient groups but all groups were significantly lower than in the controls. Levels of IgA and IgM milk-specific antibodies were similar in patients and controls. The results indicate that different immunopathogenic mechanisms are operative in these subgroups of patients with cow's milk allergy.

The pattern of the humoral-immune response to cow's milk was examined in children with eczema and in children who had acute urticarial and/or angioedematous reactions to cow's milk. Each patient group was compared to a group of age-matched controls. Whereas the eczematous patients had significantly elevated IgG, IgA and IgE milk-antibodies, patients, allergic to cow's milk, had elevated IgE milk-antibodies. These differences in the pattern of antibody response to cow's milk suggests that these two patient groups constitute separate allergic populations, and that different pathogenic mechanisms may be operative in these two skin diseases associated with elevated levels of IgE antibodies to cow's milk.

The phenomenon that large amounts of antigen, such as are absorbed during the neonatal period, suppress the IgE response while low-dose exposure enhances it was investigated by analysing the antibody responses of infants allergic to milk according to their degree of exposure to cows'-milk protein. IgG, IgA, and IgM milk-specific antibodies in these infants and in age-matched controls were measured by enzyme-linked immunosorbent assay. Milk-specific IgE and total IgE were also measured. Children allergic to milk who were breast fed and had had minimal exposure to cows' milk had decreased titres of IgG, IgA, and IgM milk antibodies compared with infants allergic to milk who, before diagnosis, had been fed substantial volumes of cows' milk. Conversely, the infants with minimal exposure to cows' milk showed vastly increased total and milk-specific IgE antibodies compared with the milk-fed infants. These results support recent experimental evidence that appreciable amounts of allergen suppress rather than stimulate IgE production. These data may have important implications for dietary regimens in at-risk infants. The results also lend support for the role of IgE in immediate-type allergic reactions and suggest that various non-IgE immune mechanisms play a part in the aetiology of intolerance to cows'-milk protein in some children.

In order to test the hypothesis that rotavirus infection leads to IgE hypersensitivity to cows' milk, evidence of past rotavirus infection, as reflected by the presence of antibodies, was sought in two groups of children with cows' milk allergy. The groups were defined according to the time to react to milk challenge. In group I there were fifteen children with an adverse reaction within 40 min of milk challenge. The twenty-one children in group II had gastrointestinal reactions between 1 hr and 24 hr after milk challenge. The results showed a higher incidence of antibodies to rotavirus in group II than group I (P less than 0.05). Although group I patients had significantly higher levels of total and milk-specific IgE antibodies to cows' milk than those in group II, both patient groups had lower levels of serum IgG, IgA and IgM than a control population. No association between the presence of rotavirus antibodies and IgE hypersensitivity to cows' milk was found. However, an association between non-IgE milk enteropathy and previous rotavirus antibodies was demonstrated.

In a study of the manifestations of cow milk allergy in 100 young children (mean age 16 months), 30 items of historical data and information relating to the effects of a standardized milk challenge were entered into a computer data base. Three clusters of patients were derived using a K-means algorithm. In group 1 were 27 patients with predominantly urticarial and angioedematous eruptions, which developed within 45 minutes of ingesting cow milk. They had positive skin test reactions to milk and elevated total and milk specific IgE serum antibody levels. In group 2, 53 patients had pallor, vomiting, or diarrhea between 45 minutes and 20 hours after milk ingestion. These children were relatively IgA deficient. The 20 patients in group 3 had eczematous or bronchitic or diarrheal symptoms; in 17 symptoms developed more than 20 hours after commencing milk ingestion. Of the patients in group 3, only those with eczema had a positive skin test reaction and elevated IgE antibodies to milk. The patients in group 3 were the most difficult to identify clinically; they had a history of chronic ill health, and symptoms developed many hours or days after commencing milk ingestion in the challenge situation. In view of the heterogeneous clinical and immunologic findings in our patients, it is unlikely that a single laboratory test will identify cow milk allergy in all susceptible patients.

T cell derived IgE-binding factors inhibited the formation of IgE-specific rosettes on both rat basophilic leukaemic cells and lymphocytes. This was demonstrated by IgE-binding factors released from either IgE-pulsed T cells or adherent cells as well as from IgE-binding factor producing hybridomas. The different binding factors inhibited the IgE-rosettes to a different degree when tested on basophils and lymphocytes, most likely due to the higher affinity for IgE of the former cell type. The results indicate that rat basophils and mouse T cells may recognize common epitopes on the Fc portion of IgE.

This study aimed to compare the efficiency and extent of induction by monomeric versus cross-linked IgE of specific receptors for IgE on normal murine splenic T cells (Fc epsilon R-T), and to study the ability of IgE-pulsed splenic adherent cells to induce receptors for IgE on T cells. Chemically cross-linked IgE was found to be both more effective and more efficient than monomeric IgE in inducing Fc epsilon R-T as measured by the ability of IgE-pulsed T cells to form specific rosettes with IgE-sensitized trinitrophenylated sheep red blood cells (TNP-SRBC). This phenomenon was dependent on both DNA and protein synthesis, suggesting that induction caused the production of new IgE receptors. It was also found that cross-linked but not monomeric IgE-pulsed normal murine adherent cells as well as their cell-free products could actively induce significant levels of specific Fc epsilon R-T. Both cross-linked IgE-pulsed T cells and adherent cells released IgE binding factor(s). These materials were capable of specifically inhibiting the binding of IgE to rat basophilic leukaemic cells (RBL) in vitro and to rat tissue mast cells in vivo. Collectively, these data provide further evidence to suggest that polymerized forms of IgE and adherent cells play important roles in the regulation of IgE responses.