Assessing the Therapeutic Impacts of HAMLET and FOLFOX on BRAF-Mutated Colorectal Cancer: A Study of Cancer Cell Survival and Mitochondrial Dynamics In Vitro and Ex Vivo.

Background and Objectives: Colorectal cancer (CRC) is a major global health challenge. The BRAF V600E mutation, found in 8-12% of CRC patients, exacerbates this by conferring poor prognosis and resistance to therapy. Our study focuses on the efficacy of the HAMLET complex, a molecular substance derived from human breast milk, on CRC cell lines and ex vivo biopsies harboring this mutation, given its previously observed selective toxicity to cancer cells. Materials and Methods: we explored the effects of combining HAMLET with the FOLFOX chemotherapy regimen on CRC cell lines and ex vivo models. Key assessments included cell viability, apoptosis/necrosis induction, and mitochondrial function, aiming to understand the mutation-specific resistance or other cellular response mechanisms. Results: HAMLET and FOLFOX alone decreased viability in CRC explants, irrespective of the BRAF mutation status. Notably, their combination yielded a marked decrease in viability, particularly in the BRAF wild-type samples, suggesting a synergistic effect. While HAMLET showed a modest inhibitory effect on mitochondrial respiration across both mutant and wild-type samples, the response varied depending on the mutation status. Significant differences emerged in the responses of the HT-29 and WiDr cell lines to HAMLET, with WiDr cells showing greater resistance, pointing to factors beyond genetic mutations influencing drug responses. A slight synergy between HAMLET and FOLFOX was observed in WiDr cells, independent of the BRAF mutation. The bioenergetic analysis highlighted differences in mitochondrial respiration between HT-29 and WiDr cells, suggesting that bioenergetic profiles could be key in determining cellular responses to HAMLET. Conclusions: We highlight the potential of HAMLET and FOLFOX as a combined therapeutic approach in BRAF wild-type CRC, significantly reducing cancer cell viability. The varied responses in CRC cell lines, especially regarding bioenergetic and mitochondrial factors, emphasize the need for a comprehensive approach considering both genetic and metabolic aspects in CRC treatment strategies.

HAMLET a human milk protein-lipid complex induces a pro-inflammatory phenotype of myeloid cells.

HAMLET is a protein-lipid complex with a specific and broad bactericidal and tumoricidal activity, that lacks cytotoxic activity against healthy cells. In this study, we show that HAMLET also has general immune-stimulatory effects on primary human monocyte-derived dendritic cells and macrophages (Mo-DC and Mo-M) and murine RAW264.7 macrophages. HAMLET, but not its components alpha-lactalbumin or oleic acid, induces mature CD14low/- CD83+ Mo-DC and M1-like CD14+ CD86++ Mo-M surface phenotypes. Concomitantly, inflammatory mediators, including IL-2, IL-6, IL-10, IL-12 and MIP-1α, were released in the supernatant of HAMLET-stimulated cells, indicating a mainly pro-inflammatory phenotype. The HAMLET-induced phenotype was mediated by calcium, NFκB and p38 MAPK signaling in Mo-DCs and calcium, NFκB and ERK signaling in Mo-M as inhibitors of these pathways almost completely blocked the induction of mature Mo-DCs and M1-like Mo-M. Compared to unstimulated Mo-DCs, HAMLET-stimulated Mo-DCs were more potent in inducing T cell proliferation and HAMLET-stimulated macrophages were more efficient in phagocytosis of Streptococcus pneumoniae in vitro. This indicates a functionally activated phenotype of HAMLET-stimulated DCs and macrophages. Combined, we propose that HAMLET has a two-fold anti-bacterial activity; one inducing direct cytotoxic activity, the other indirectly mediating elimination of bacteria by activation of immune cells of the myeloid lineage.

The human milk protein-lipid complex HAMLET disrupts glycolysis and induces death in Streptococcus pneumoniae.

HAMLET is a complex of human α-lactalbumin (ALA) and oleic acid and kills several Gram-positive bacteria by a mechanism that bears resemblance to apoptosis in eukaryotic cells. To identify HAMLET's bacterial targets, here we used Streptococcus pneumoniae as a model organism and employed a proteomic approach that identified several potential candidates. Two of these targets were the glycolytic enzymes fructose bisphosphate aldolase (FBPA) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Treatment of pneumococci with HAMLET immediately inhibited their ATP and lactate production, suggesting that HAMLET inhibits glycolysis. This observation was supported by experiments with recombinant bacterial enzymes, along with biochemical and bacterial viability assays, indicating that HAMLET's activity is partially inhibited by high glucose-mediated stimulation of glycolysis but enhanced in the presence of the glycolysis inhibitor 2-deoxyglucose. Both HAMLET and ALA bound directly to each glycolytic enzyme in solution and solid-phase assays and effectively inhibited their enzymatic activities. In contrast, oleic acid alone had little to no inhibitory activity. However, ALA alone also exhibited no bactericidal activity and did not block glycolysis in whole cells, suggesting a role for the lipid moiety in the internalization of HAMLET into the bacterial cells to reach its target(s). This was verified by inhibition of enzyme activity in whole cells after HAMLET but not ALA exposure. The results of this study suggest that part of HAMLET's antibacterial activity relates to its ability to target and inhibit glycolytic enzymes, providing an example of a natural antimicrobial agent that specifically targets glycolysis.

A Protein Complex from Human Milk Enhances the Activity of Antibiotics and Drugs against Mycobacterium tuberculosis.

Mycobacterium tuberculosis, the causative agent of human tuberculosis (TB), has surpassed HIV/AIDS as the leading cause of death from a single infectious agent. The increasing occurrence of drug-resistant strains has become a major challenge for health care systems and, in some cases, has rendered TB untreatable. However, the development of new TB drugs has been plagued with high failure rates and costs. Alternative strategies to increase the efficacy of current TB treatment regimens include host-directed therapies or agents that make M. tuberculosis more susceptible to existing TB drugs. In this study, we show that HAMLET, an α-lactalbumin-oleic acid complex derived from human milk, has bactericidal activity against M. tuberculosis HAMLET consists of a micellar oleic acid core surrounded by a shell of partially denatured α-lactalbumin molecules and unloads oleic acid into cells upon contact with lipid membranes. At sublethal concentrations, HAMLET potentiated a remarkably broad array of TB drugs and antibiotics against M. tuberculosis For example, the minimal inhibitory concentrations of rifampin, bedaquiline, delamanid, and clarithromycin were decreased by 8- to 16-fold. HAMLET also killed M. tuberculosis and enhanced the efficacy of TB drugs inside macrophages, a natural habitat of M. tuberculosis Previous studies showed that HAMLET is stable after oral delivery in mice and nontoxic in humans and that it is possible to package hydrophobic compounds in the oleic acid core of HAMLET to increase their solubility and metabolic stability. The potential of HAMLET and other liprotides as drug delivery and sensitization agents in TB chemotherapy is discussed here.

Topographical memory analyzed in mice using the Hamlet test, a novel complex maze.

The Hamlet test is an innovative device providing a complex environment for testing topographic memory in mice. Animals were trained in groups for weeks in a small village with a central agora, streets expanding from it towards five functionalized houses, where they can drink, eat, hide, run, interact with a stranger mouse. Memory was tested by depriving mice from water or food and analyzing their ability to locate the Drink/Eat house. Exploration and memory were analyzed in different strains, gender, and after different training periods and delays. After 2 weeks training, differences in exploration patterns were observed between strains, but not gender. Neuroanatomical structures activated by training, identified using FosB/ΔFosB immunolabelling, showed an involvement of the hippocampus-subiculum-parahippocampal gyrus axis and dopaminergic structures. Training increased hippocampal neurogenesis (cell proliferation and neuronal maturation) and modified the amnesic efficacy of muscarinic or nicotinic cholinergic antagonists. Moreover, topographical disorientation in Alzheimer's disease was addressed using intracerebroventricular injection of amyloid ß25-35 peptide in trained mice. When retested after 7 days, Aß25-35-treated mice showed memory impairment. The Hamlet test specifically allows analysis of topographical memory in mice, based on complex environment. It offers an innovative tool for various ethological or pharmacological research needs. For instance, it allowed to examine topographical disorientation, a warning sign in Alzheimer's disease.

Hamlet and psychiatry intertwined.

OBJECTIVES:: This article considers selected landmarks in the history of psychiatry and their impact on Hamlet productions, including Burton's Anatomy of Melancholia, Emil Kraepelin's manic-depression, Freud's oedipal complex and R.D. Laing's 'divided self'. Additionally, this article considers the way Shakespeare's Hamlet has influenced the course of psychiatry. CONCLUSION:: The linkages between psychiatry and Hamlet have existed since the 17th century, and perhaps Shakespeare's Hamlet should have a place on every psychiatrist's shelf.

HAMLET - A protein-lipid complex with broad tumoricidal activity.

HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) is a tumoricidal protein-lipid complex with broad effects against cancer cells of different origin. The therapeutic potential is emphasized by a high degree of specificity for tumor tissue. Here we review early studies of HAMLET, in collaboration with the Orrenius laboratory, and some key features of the subsequent development of the HAMLET project. The early studies focused on the apoptotic response that accompanies death in HAMLET treated tumor cells and the role of mitochondria in this process. In subsequent studies, we have identified a sequence of interactions that starts with the membrane integration of HAMLET and the activation of ion fluxes followed by HAMLET internalization, progressive inhibition of MAPK kinases and GTPases and sorting of HAMLET to different cellular compartments, including the nuclei. Therapeutic efficacy of HAMLET has been demonstrated in animal models of glioblastoma, bladder cancer and intestinal cancer. In clinical studies, HAMLET has been shown to target skin papillomas and bladder cancers. The findings identify HAMLET as a new drug candidate with promising selectivity for cancer cells and a strong therapeutic potential.

Tailoring thermal treatment to form liprotide complexes between oleic acid and different proteins.

Liprotides are protein-lipid complexes in which the fatty acids form a micelle-like core surrounded by a shell of partially unfolded protein molecules. These complexes can be formed in different ways. The simplest approach is a thermal treatment where protein and fatty acid are mixed and then incubated at elevated temperatures. Using this approach we here demonstrate that we can monitor liprotide formation in real time using Small-Angle X-ray Scattering (SAXS). Optimal conditions for liprotide formation, i.e. temperature and incubation times, as well as liprotide stability and structure, vary for different proteins. The apo form of α-lactalbumin (aLA) forms liprotides at room temperature, however, Ovalbumin (Ova) and Bovine Serum Albumin (BSA) require elevated temperatures (≥60°C) to form liprotides, and in addition, they need to be returned to lower temperatures to remain stable; repeated cycles of heating and cooling gradually dissociate the liprotides in parallel with the formation of disulfide-bonded aggregates. Real-time tracking of the formation of liprotides of BSA or Ova with OA at 60-65°C showed that liprotide formation takes place within a period of 12-18min and is preceded by a loss of secondary structure of the protein and binding of OA to the protein. Our SAXS-based approach provides a straightforward strategy to optimize liprotide formation for a wide range of different proteins.

Oleic acid complex of bovine α-lactalbumin induces eryptosis in human and other erythrocytes by a Ca(2+)-independent mechanism.

BACKGROUND: Complexes of oleic acid (OA) with milk α-lactalbumin, received remarkable attention in view of their selective toxicity towards a spectrum of tumors during the last two decades. OA complexes of some structurally related/unrelated proteins are also tumoricidal. Erythrocytes are among the few differentiated cells that are sensitive and undergo hemolysis when exposed to the complexes. METHODS: The effects of OA complex of bovine α-lactalbumin (Bovine Alpha-lactalbumin Made LEthal to Tumor cells, BAMLET) on human, goat and chicken erythrocytes on calcein leakage, phosphatidylserine exposure, morphological changes and hemolysis were studied by confocal microscopy, FACS analysis, scanning electron microscopy and measuring hemoglobin release. RESULTS: Erythrocytes exposed to BAMLET undergo eryptosis-like alterations as revealed by calcein leakage, surface phosphatidylserine exposure and transformation to echinocytes at low concentrations and hemolysis when the concentration of the complex was raised. Ca(2+) was not essential and restricted the alterations when included in the medium. The BAMLET-induced alterations in human erythrocytes were prevented by the cation channel inhibitors, amiloride and BaCl2 but not by inhibitors of thiol proteases, sphingomyelinase and by the antioxidant N-acetyl cysteine. CONCLUSIONS: The work shows for the first time that low concentrations of BAMLET induces eryptosis in erythrocytes by a novel mechanism not requiring Ca(2+) and hemolysis by detergent-like action by the released OA at higher concentrations. GENERAL SIGNIFICANCE: The study points out to the need for a comprehensive evaluation of the toxicity of OA complexes of α-lactalbumin and other proteins towards erythrocytes and other differentiated cells before being considered for therapy.

A Characeae Cells Plasma Membrane as a Model for Selection of Bioactive Compounds and Drugs: Interaction of HAMLET-Like Complexes with Ion Channels of Chara corallina Cells Plasmalemma.

Interaction of a HAMLET-like La-OA cytotoxic complex (human α-lactalbumin-oleic acid) and its constituents with the excitable plasmalemma of giant Chara corallina cells was investigated. The voltage-clamp technique was used to study Ca2+ and Cl- transient currents in the plasmalemma of intact cells. The action of the complex and OA on the target cell membrane has a dose-dependent character. It was found that the La-OA complex has an inhibiting effect on Ca2+ current across the plasmalemma, while α-lactalbumin alone does not affect the electrophysiological characteristics of the cellular membrane. However, oleic acid blocks Ca2+ current across the plasmalemma. This is accompanied by the induction of a non-selective conductivity in the cellular membrane, a decrease in the resting potential and plasma membrane resistance of algal cells. We propose that the cytotoxicity of La-OA and other HAMLET-like complexes is determined by oleic acid acting as a blocker of potential-dependent Ca2+ channels in the plasma membrane of target cells. The presented results show that the study model of green algae C. corallina cells plasmalemma is a convenient tool for the investigation of ion channels in many animal cells.

The targeted proteins in tumor cells treated with the α-lactalbumin-oleic acid complex examined by descriptive and quantitative liquid chromatography-tandem mass spectrometry.

An α-lactalbumin-oleic acid (α-LA-OA) complex has exhibited selective antitumor activity in animal models and clinical trials. Although apoptosis and autophagy are activated and the functions of several organelles are disrupted in response to α-LA-OA, the detailed antitumor mechanism remains unclear. In this study, we used a novel technique, isobaric tags for relative and absolute quantitation, to analyze the proteome of tumor cells treated with α-LA-OA. We identified 112 differentially expressed proteins: 95 were upregulated to satisfy the metabolism of tumor cells; 17 were downregulated and targets of α-LA-OA. According to the differentially expressed proteins, α-LA-OA exerted its antitumor activity by disrupting cytoskeleton stability and cell motility, and by inhibiting DNA, lipid, and ATP synthesis, leading to cellular stress and activation of programmed cell death. This study provides a systematic evaluation of the antitumor activity of α-LA-OA, identifying its interacting targets and establishing the theoretical basis of α-LA-OA for use in cancer therapy.

Bovine lactoferrin binds oleic acid to form an anti-tumor complex similar to HAMLET.

α-Lactalbumin (α-LA) can bind oleic acid (OA) to form HAMLET-like complexes, which exhibited highly selective anti-tumor activity in vitro and in vivo. Considering the structural similarity to α-LA, we conjectured that lactoferrin (LF) could also bind OA to obtain a complex with anti-tumor activity. In this study, LF-OA was prepared and its activity and structural changes were compared with α-LA-OA. The anti-tumor activity was evaluated by methylene blue assay, while the apoptosis mechanism was analyzed using flow cytometry and Western blot. Structural changes of LF-OA were measured by fluorescence spectroscopy and circular dichroism. The interactions of OA with LF and α-LA were evaluated by isothermal titration calorimetry (ITC). LF-OA was obtained by heat-treatment at pH8.0 with LD50 of 4.88, 4.95 and 4.62µM for HepG2, HT29, and MCF-7 cells, respectively, all of which were 10 times higher than those of α-LA-OA. Similar to HAMLET, LF-OA induced apoptosis in tumor cells through both death receptor- and mitochondrial-mediated pathways. Exposure of tryptophan residues and the hydrophobic regions as well as the loss of tertiary structure were observed in LF-OA. Besides these similarities, LF showed different secondary structure changes when compared with α-LA, with a decrease of α-helix and ß-turn and an increase of ß-sheet and random coil. ITC results showed that there was a higher binding number of OA to LF than to α-LA, while both of the proteins interacted with OA through van der Waals forces and hydrogen bonds. This study provides a theoretical basis for further exploration of protein-OA complexes.

Lipids as tumoricidal components of human α-lactalbumin made lethal to tumor cells (HAMLET): unique and shared effects on signaling and death.

Long-chain fatty acids are internalized by receptor-mediated mechanisms or receptor-independent diffusion across cytoplasmic membranes and are utilized as nutrients, building blocks, and signaling intermediates. Here we describe how the association of long-chain fatty acids to a partially unfolded, extracellular protein can alter the presentation to target cells and cellular effects. HAMLET (human α-lactalbumin made lethal to tumor cells) is a tumoricidal complex of partially unfolded α-lactalbumin and oleic acid (OA). As OA lacks independent tumoricidal activity at concentrations equimolar to HAMLET, the contribution of the lipid has been debated. We show by natural abundance (13)C NMR that the lipid in HAMLET is deprotonated and by chromatography that oleate rather than oleic acid is the relevant HAMLET constituent. Compared with HAMLET, oleate (175 µm) showed weak effects on ion fluxes and gene expression. Unlike HAMLET, which causes metabolic paralysis, fatty acid metabolites were less strongly altered. The functional overlap increased with higher oleate concentrations (500 µm). Cellular responses to OA were weak or absent, suggesting that deprotonation favors cellular interactions of fatty acids. Fatty acids may thus exert some of their essential effects on host cells when in the deprotonated state and when presented in the context of a partially unfolded protein.

Molecular mechanisms of the cytotoxicity of human α-lactalbumin made lethal to tumor cells (HAMLET) and other protein-oleic acid complexes.

Although HAMLET (human α-lactalbumin made lethal to tumor cells), a complex formed by human α-lactalbumin and oleic acid, has a unique apoptotic activity for the selective killing of tumor cells, the molecular mechanisms of expression of the HAMLET activity are not well understood. Therefore, we studied the molecular properties of HAMLET and its goat counterpart, GAMLET (goat α-lactalbumin made lethal to tumor cells), by pulse field gradient NMR and 920-MHz two-dimensional NMR techniques. We also examined the expression of HAMLET-like activities of complexes between oleic acid and other proteins that form a stable molten globule state. We observed that both HAMLET and GAMLET at pH 7.5 were heterogeneous, composed of the native protein, the monomeric molten globule-like state, and the oligomeric species. At pH 2.0 and 50 °C, HAMLET and GAMLET appeared in the monomeric state, and we identified the oleic acid-binding site in the complexes by two-dimensional NMR. Rather surprisingly, the binding site thus identified was markedly different between HAMLET and GAMLET. Furthermore, canine milk lysozyme, apo-myoglobin, and ß2-microglobulin all formed the HAMLET-like complex with the anti-tumor activity, when the protein was treated with oleic acid under conditions in which their molten globule states were stable. From these results, we conclude that the protein portion of HAMLET, GAMLET, and the other HAMLET-like protein-oleic acid complexes is not the origin of their cytotoxicity to tumor cells and that the protein portion of these complexes plays a role in the delivery of cytotoxic oleic acid molecules into tumor cells across the cell membrane.

Cytotoxicity of bovine α-lactalbumin: oleic acid complexes correlates with the disruption of lipid membranes.

HAMLET/BAMLET (Human/Bovine α-Lactalbumin Made Lethal to Tumors) is a tumoricidal substance composed of partially unfolded human/bovine α-lactalbumin (HLA/BLA) and several oleic acid (OA) molecules. The HAMLET mechanism of interaction involves an insufficiently understood effect on the membrane or its embedded components. We examined the effect of BLAOA (bovine α-lactalbumin complexed with oleic acid, a HAMLET-like substance) and its individual components on cells and artificial lipid membranes using viability staining and metabolic dyes, fluorescence spectroscopy, leakage integrity assays and microscopy. Our results show a dose-dependency of OA used to prepare BLAOA on its ability to induce tumor cell death, and a correlation between leakage and cell death. BLAOA incorporates into the membrane, tightens the lipid packing and lowers their solvent accessibility. Fluorescence imaging reveals that giant unilamellar vesicles (GUVs) develop blebs and eventually collapse upon exposure to BLAOA, indicating that the lipid packing reorganization can translate into observable morphological effects. These effects are observed to be local in GUVs, and a tightly packed and solvent-shielded lipid environment is associated with leakage and GUV disruption. Furthermore, the effects of BLAOA on membrane are pH dependent, with an optimum of activity on artificial membranes near neutral pHs. While BLA alone is effective at membrane disruption at acidic pHs, OA is ineffective in a pH range of 4.5 to 9.1. Taken together, this supports a model where the lipid, fatty acid and protein components enhance each other's ability to affect the overall integrity of the membrane.

Small-angle X-ray scattering of BAMLET at pH 12: a complex of α-lactalbumin and oleic acid.

BAMLET (Bovine Alpha-lactalbumin Made LEthal to Tumors) is a member of the family of the HAMLET-like complexes, a novel class of protein-based anti-cancer complexes that incorporate oleic acid and deliver it to cancer cells. Small angle X-ray scattering (SAXS) was performed on the complex at pH 12, examining the high pH structure as a function of oleic acid added. The SAXS data for BAMLET species prepared with a range of oleic acid concentrations indicate extended, irregular, partially unfolded protein conformations that vary with the oleic acid concentration. Increases in oleic acid concentration correlate with increasing radius of gyration without an increase in maximum particle dimension, indicating decreasing protein density. The models for the highest oleic acid content BAMLET indicate an unusual coiled elongated structure that contrasts with apo-α-lactalbumin at pH 12, which is an elongated globular molecule, suggesting that oleic acid inhibits the folding or collapse of the protein component of BAMLET to the globular form. Circular dichroism of BAMLET and apo-α-lactalbumin was performed and the results suggest that α-lactalbumin and BAMLET unfold in a continuum of increasing degree of unfolded states. Taken together, these results support a model in which BAMLET retains oleic acid by non-specific association in the core of partially unfolded protein, and represent a new type of lipoprotein structure.

HAMLET, a human milk protein-lipid complex, modulates amoxicillin induced changes in an ex vivo biofilm model of the oral microbiome.

Challenges from infections caused by biofilms and antimicrobial resistance highlight the need for novel antimicrobials that work in conjunction with antibiotics and minimize resistance risk. In this study we investigated the composite effect of HAMLET (human alpha-lactalbumin made lethal to tumor cells), a human milk protein-lipid complex and amoxicillin on microbial ecology using an ex vivo oral biofilm model with pooled saliva samples. HAMLET was chosen due to its multi-targeted antimicrobial mechanism, together with its synergistic effect with antibiotics on single species pathogens, and low risk of resistance development. The combination of HAMLET and low concentrations of amoxicillin significantly reduced biofilm viability, while each of them alone had little or no impact. Using a whole metagenomics approach, we found that the combination promoted a remarkable shift in overall microbial composition compared to the untreated samples. A large proportion of the bacterial species in the combined treatment were Lactobacillus crispatus, a species with probiotic effects, whereas it was only detected in a minor fraction in untreated samples. Although resistome analysis indicated no major shifts in alpha-diversity, the results showed the presence of TEM beta-lactamase genes in low proportions in all treated samples but absence in untreated samples. Our study illustrates HAMLET's capability to alter the effects of amoxicillin on the oral microbiome and potentially favor the growth of selected probiotic bacteria when in combination. The findings extend previous knowledge on the combined effects of HAMLET and antibiotics against target pathogens to include potential modulatory effects on polymicrobial biofilms of human origin.

Beyond Milk and Nurture: Breastfeeding's Powerful Impact on Breast Cancer.

Breast cancer (BC) stands as a global concern, given its high incidence and impact on women's mortality. This complex disease has roots in various risk factors, some modifiable and others not. Understanding and identifying these factors can be instrumental in both preventing BC and improving survival rates. Remarkably, women's reproductive behaviors have emerged as critical determinants of BC susceptibility. Numerous studies have shed light on how aspects including age of menarche, first pregnancy and menopause along with number of pregnancies, hormone replacement therapies, can influence one's risk of developing BC. Furthermore, the act of breastfeeding and its duration have shown an inverse relationship with BC risk. This review delves into the biological and molecular mechanisms associated with breastfeeding that contribute to BC protection. It highlights the role of endocrine processes triggered by suckling stimulation, the gradual onset of lactational amenorrhea, delayed weaning, reduced lifetime menstrual cycles, chromosomal repair mechanisms, and immunological events throughout the lactation cycle. These insights provide a potential explanation for the protective effects conferred by breastfeeding against breast carcinomas.

Mourning in Hamlet: Turning ancestral ghosts into ancestors.

Extracts from Shakespeare's Hamlet are used to show how obstacles to mourning may arise from the persistent demands of melancholic internal objects demanding repair and revenge. It is only with the development of symbolic function as a result of separateness between self and object that reparation becomes possible and ghosts are turned into ancestors.

Treatment of Mouse Infants with Amoxicillin, but Not the Human Milk-Derived Antimicrobial HAMLET, Impairs Lung Th17 Responses.

Emerging evidence suggests differential effects of therapeutic antibiotics on infant T cell responses to pathogens. In this study, we explored the impact of the treatment of mouse infants with amoxicillin and the human milk-derived antimicrobial HAMLET (human alpha-lactalbumin made lethal to tumor cells) on T cell responses to Streptococcus pneumoniae. Lung cells and splenocytes were isolated from the infant mice subjected to intranasal administration of amoxicillin, HAMLET, or a combination of HAMLET and amoxicillin, and cultured with S. pneumoniae to measure T cell responses. After in-vitro stimulation with S. pneumoniae, lung cells from amoxicillin- or amoxicillin plus HAMLET-treated mice produced lower levels of Th17 (IL-17A), but not Th1 (IFN-γ), cytokine than mice receiving HAMLET or PBS. IL-17A/IFN-γ cytokine levels produced by the stimulated splenocytes, on the other hand, revealed no significant difference among treatment groups. Further analysis of T cell cytokine profiles by flow cytometry showed that lung CD4+, but not CD8+, T cells from amoxicillin- or HAMLET plus amoxicillin-treated mice expressed decreased levels of IL-17A compared to those from HAMLET-exposed or control mice. Collectively, these results indicate that exposure of infant mice to amoxicillin, but not HAMLET, may suppress lung Th17 responses to S. pneumoniae.