NEW VISTAS IN PSYCHOSOMATIC MEDICINE

Further understanding in the field of psychosomatic medicine has come to light recently as the result of new approaches and methods of research.Such diseases as hypertension, ulcerative colitis, rheumatoid arthritis, peptic ulcer, diabetes and cardiovascular dysfunction may represent the body''s method of adapting to chronic stress, according to Selye''s concept of the general adaptation syndrome, with the phases of alarm, resistance and exhaustion.It has been postulated that unconscious dynamics of which patients are unaware are crucial in the understanding and interpretation of physiological research and therapy of patients with psychosomatic disorders.The concept of partial regression was applicable to patients with psychosomatic illness who were highly successful in social, economic and professional spheres. The illness was viewed as a protection against psychological regression by limited somatic regression.Pilot studies suggested that patients seriously ill with such disorders as ulcerative colitis and asthma responded favorably to enforced psychological regression and exploitation of dependency by excessive coddling, babying and mothering by an “all-giving” physician in a hospital setting.Good physician-patient relationship remains the keystone in therapy and is the common denominator to many so-called successful modes of treatment.     

Anatomic Characteristics Associated with Head Splitting in Cabbage (Brassica oleracea var. capitata L.)

Cabbage belonging to Brassicaceae family is one of the most important vegetables cultivated worldwide. The economically important part of cabbage crop is head, formed by leaves which may be of splitting and non-splitting types. Cabbage varieties showing head splitting causes huge loss to the farmers and therefore finding the molecular and structural basis of splitting types would be helpful to breeders. To determine which anatomical characteristics were related to head-splitting in cabbage, we analyzed two contrasting cabbage lines and their offspring using a field emission scanning electron microscope. The inbred line “747” is an early head-splitting type, while the inbred line “748” is a head-splitting-resistant type. The petiole cells of “747” seems to be larger than those of “748” at maturity; however, there was no significant difference in petiole cell size at both pre-heading and maturity stages. The lower epidermis cells of “747” were larger than those of “748” at the pre-heading and maturity stages. “747” had thinner epidermis cell wall than “748” at maturity stage, however, there was no difference of the epidermis cell wall thickness in the two lines at the pre-heading stage. The head-splitting plants in the F₁ and F₂ population inherited the larger cell size and thinner cell walls of epidermis cells in the petiole. In the petiole cell walls of “747” and the F₁ and F₂ plants that formed splitting heads, the cellulose microfibrils were loose and had separated from each other. These findings verified that anomalous cellulose microfibrils, larger cell size and thinner-walled epidermis cells are important genetic factors that make cabbage heads prone to splitting.     

Mitochondrial biogenesis in epithelial cancer cells promotes breast cancer tumor growth and confers autophagy resistance

Here, we set out to test the novel hypothesis that increased mitochondrial biogenesis in epithelial cancer cells would “fuel” enhanced tumor growth. For this purpose, we generated MDA-MB-231 cells (a triple-negative human breast cancer cell line) overexpressing PGC-1α and MitoNEET, which are established molecules that drive mitochondrial biogenesis and increased mitochondrial oxidative phosphorylation (OXPHOS). Interestingly, both PGC-1α and MitoNEET increased the abundance of OXPHOS protein complexes, conferred autophagy resistance under conditions of starvation and increased tumor growth by up to ~3-fold. However, this increase in tumor growth was independent of neo-angiogenesis, as assessed by immunostaining and quantitation of vessel density using CD31 antibodies. Quantitatively similar increases in tumor growth were also observed by overexpression of PGC-1β and POLRMT in MDA-MB-231 cells, which are also responsible for mediating increased mitochondrial biogenesis. Thus, we propose that increased mitochondrial “power” in epithelial cancer cells oncogenically promotes tumor growth by conferring autophagy resistance. As such, PGC-1α, PGC-1β, mitoNEET and POLRMT should all be considered as tumor promoters or “metabolic oncogenes.” Our results are consistent with numerous previous clinical studies showing that metformin (a weak mitochondrial “poison”) prevents the onset of nearly all types of human cancers in diabetic patients. Therefore, metformin (a complex I inhibitor) and other mitochondrial inhibitors should be developed as novel anticancer therapies, targeting mitochondrial metabolism in cancer cells.     

Traumatic Rupture of the Spleen with Delayed Intraperitoneal Hemorrhage During Pregnancy

The extent of anatomical involvement (rectosigmoid, left colon, entire colon, segmental, and enterocolon) and clinical behaviour (acute fulminating, recurrent colitis, and chronic continuous colitis) must be considered in assessing the prognosis and management of patients with ulcerative colitis. The severity of symptoms and signs is highly variable. It ranges from the virtually asymptomatic patient with mild proctitis to the patient with acute fulminating colitis with arthritis, skin lesions and “toxic megacolon” who constitutes a medical emergency. Consequently, there is no single therapeutic formula to be applied in this disease. Treatment is reviewed with reference to the recent literature.     

THE USE OF BANTHINE IN THE TREATMENT OF DIGESTIVE DISTURBANCES

Banthine® was used in the treatment of patients with various diseases, organic and functional, of the gastrointestinal tract. Good response was obtained in a high proportion of cases of duodenal, stomal and gastric ulcer, and of hypertrophic gastritis. In some instances, patients who did not have good response at first were relieved later when the size of doses and the dosage schedule were adjusted to fit their particular needs.Some patients “felt so well” during Banthine therapy that they departed from prescribed diet and violated injunctions against use of alcohol and tobacco, and symptoms recurred.Nine patients with history of recurrent bouts of pain from ulcer for several years took small doses of Banthine constantly, or occasionally at times of stress, as a prophylactic measure after the symptoms were relieved by therapeutic doses. None of them had recurrence while following the prophylactic regimen.In most of the cases of peptic ulcer in which the response was recorded as “poor,” it was because distressing side-effects dictated discontinuance of the drug. Several elderly male patients had severe urinary retention. Paralytic ileus developed postoperatively in one patient who was receiving Banthine. Less severe side reactions—dry mouth, blurring of vision, urinary slowing — were for the most part transient.Few patients with functional indigestion, chronic non-specific colitis or regional enteritis were relieved. Most of the patients with functional indigestion reported exacerbation of symptoms when Banthine was given. This was believed to be based on emotional reaction to the hypomotility induced by the drug.     

Salmonella enterica serovar typhimurium exploits inflammation to compete with the intestinal microbiota

Most mucosal surfaces of the mammalian body are colonized by microbial communities (“microbiota”). A high density of commensal microbiota inhabits the intestine and shields from infection (“colonization resistance”). The virulence strategies allowing enteropathogenic bacteria to successfully compete with the microbiota and overcome colonization resistance are poorly understood. Here, we investigated manipulation of the intestinal microbiota by the enteropathogenic bacterium Salmonella enterica subspecies 1 serovar Typhimurium (S. Tm) in a mouse colitis model: we found that inflammatory host responses induced by S. Tm changed microbiota composition and suppressed its growth. In contrast to wild-type S. Tm, an avirulent invGsseD mutant failing to trigger colitis was outcompeted by the microbiota. This competitive defect was reverted if inflammation was provided concomitantly by mixed infection with wild-type S. Tm or in mice (IL10^−/−, VILLIN-HA^CL4-CD8) with inflammatory bowel disease. Thus, inflammation is necessary and sufficient for overcoming colonization resistance. This reveals a new concept in infectious disease: in contrast to current thinking, inflammation is not always detrimental for the pathogen. Triggering the host's immune defence can shift the balance between the protective microbiota and the pathogen in favour of the pathogen.     

A “Prime-Pull” Vaccine Strategy Has a Modest Effect on Local and Systemic Antibody Responses to HIV gp140 in Mice

One potential strategy for the prevention of HIV infection is to induce virus specific mucosal antibody that can act as an immune barrier to prevent transmission. The mucosal application of chemokines after immunisation, termed “prime-pull”, has been shown to recruit T cells to mucosal sites. We wished to determine whether this strategy could be used to increase B cells and antibody in the vaginal mucosa following immunisation with an HIV antigen. BALB/c mice were immunised intranasally with trimeric gp140 prior to vaginal application of the chemokine CCL28 or the synthetic TLR4 ligand MPLA, without antigen six days later. There was no increase in vaginal IgA, IgG or B cells following the application of CCL28, however vaginal application of MPLA led to a significant boost in antigen specific vaginal IgA. Follow up studies to investigate the effect of the timing of the “pull” stimulation demonstrated that when given 14 days after the initial immunisation MPLA significantly increased systemic antibody responses. We speculate that this may be due to residual inflammation prior to re-immunisation. Overall we conclude that in contrast to the previously observed effect on T cells, the use of “prime-pull” has only a modest effect on B cells and antibody.     

Microdiverticula and submucosal epithelial elements in ulcerative and granulomatous diseases of the ileum and colon

A case of granulomatous ileocolitis which also showed features of ulcerative colitis, and in which lesions were believed to be due to inflammation of previously undescribed structures, microdiverticula, was reported previously. Subsequently, sections of the segments of bowel received from 17 cases of regional enteritis and 16 cases of ulcerative colitis were re-examined for the presence of similar microdiverticula and submucosal epithelial elements. The same structures were present in five cases of regional enteritis and in 14 of ulcerative colitis. A granulomatous type of inflammatory response was found in the bowel and lymph nodes in 14 of the 17 cases of regional enteritis, and in none of the cases labelled ulcerative colitis. In spite of this, difficulty was experienced in placing some cases in one or other diagnostic category. This re-emphasizes the possibility that the two conditions may not be etiologically discrete, and that both may be, in fact, related to the presence of microdiverticula. In addition, it may be that colitis cystica profunda is a result of microdiverticulosis in which the submucosal glandular structures attain unusually large proportions.     

Milk fat globule-epidermal growth factor 8 is decreased in intestinal epithelium of ulcerative colitis patients and thereby causes increased apoptosis and impaired wound healing

Milk fat globule-epidermal growth factor 8 (MFG-E8) plays an important role in maintaining intestinal barrier homeostasis and accelerating intestinal restitution. However, studies of MFG-E8 expression in humans with ulcerative colitis are lacking. We examined MFG-E8 expression in colonic mucosal biopsies from ulcerative colitis patients and healthy controls (n = 26 each) by real-time quantitative polymerase chain reaction (PCR), Western blot analysis and immunohistochemistry. MFG-E8 mRNA and protein expression was lower in ulcerative colitis patients than in controls. MFG-E8 expression was inversely correlated with mucosal inflammatory activity and clinical disease activity in patients. MFG-E8 was present in human intestinal epithelial cells both in vivo and in vitro. Apoptosis induction was also detected in the intestinal epithelium of ulcerative colitis patients by terminal-deoxynucleoitidyl transferase mediated nick-end labeling assay. We used lentiviral vectors encoding human MFG-E8 targeting short hairpin RNA to obtain MFG-E8 knockdown intestinal epithelia cell clones. MFG-E8 knockdown could promote apoptosis in intestinal epithelial cell lines, accompanied by a decrease in level of the antiapoptotic protein B-cell lymphoma 2 (BCL-2) and induction of the proapoptotic protein BCL2-associated protein X (BAX). The addition of recombinant human MFG-E8 led to decreased BAX and cleaved caspase-3 levels and induction of BCL-2 level in intestinal epithelia cells. MFG-E8 knockdown also attenuated wound healing on scratch assay of intestinal epithelial cells. The mRNA level of intestinal trefoid factor 3, a pivotal factor in intestinal epithelial cell migration and restitution, was downregulated with MFG-E8 knockdown. In conclusion, we demonstrated that decreased colonic MFG-E8 expression in patients with ulcerative colitis may be associated with mucosal inflammatory activity and clinical disease activity through basal cell apoptosis and preventing tissue healing in the pathogenesis of ulcerative colitis.     

Epithelial cell plasticity: breaking boundaries and changing landscapes

Epithelial tissues respond to a wide variety of environmental and genotoxic stresses. As an adaptive mechanism, cells can deviate from their natural paths to acquire new identities, both within and across lineages. Under extreme conditions, epithelial tissues can utilize “shape‐shifting” mechanisms whereby they alter their form and function at a tissue‐wide scale. Mounting evidence suggests that in order to acquire these alternate tissue identities, cells follow a core set of “tissue logic” principles based on developmental paradigms. Here, we review the terminology and the concepts that have been put forward to describe cell plasticity. We also provide insights into various cell intrinsic and extrinsic factors, including genetic mutations, inflammation, microbiota, and therapeutic agents that contribute to cell plasticity. Additionally, we discuss recent studies that have sought to decode the “syntax” of plasticity—i.e., the cellular and molecular principles through which cells acquire new identities in both homeostatic and malignant epithelial tissues—and how these processes can be manipulated for developing novel cancer therapeutics.     

The autophagic tumor stroma model of cancer or “battery-operated tumor growth”: A simple solution to the autophagy paradox

The role of autophagy in tumorigenesis is controversial. Both autophagy inhibitors (chloroquine) and autophagy promoters (rapamycin) block tumorigenesis by unknown mechanism(s). This is called the “Autophagy Paradox.” We have recently reported a simple solution to this paradox. We demonstrated that epithelial cancer cells use oxidative stress to induce autophagy in the tumor microenvironment. As a consequence, the autophagic tumor stroma generates recycled nutrients that can then be used as chemical building blocks by anabolic epithelial cancer cells. This model results in a net energy transfer from the tumor stroma to epithelial cancer cells (an energy imbalance), thereby promoting tumor growth. This net energy transfer is both unilateral and vectorial, from the tumor stroma to the epithelial cancer cells, representing a true host-parasite relationship. We have termed this new paradigm “The Autophagic Tumor Stroma Model of Cancer Cell Metabolism” or “Battery-Operated Tumor Growth.” In this sense, autophagy in the tumor stroma serves as a “battery” to fuel tumor growth, progression and metastasis, independently of angiogenesis. Using this model, the systemic induction of autophagy will prevent epithelial cancer cells from using recycled nutrients, while the systemic inhibiton of autophagy will prevent stromal cells from producing recycled nutrients—both effectively “starving” cancer cells. We discuss the idea that tumor cells could become resistant to the systemic induction of autophagy by the upregulation of natural, endogenous autophagy inhibitors in cancer cells. Alternatively, tumor cells could also become resistant to the systemic induction of autophagy by the genetic silencing/deletion of pro-autophagic molecules, such as Beclin1. If autophagy resistance develops in cancer cells, then the systemic inhibition of autophagy would provide a therapeutic solution to this type of drug resistance, as it would still target autophagy in the tumor stroma. As such, an anti-cancer therapy that combines the alternating use of both autophagy promoters and autophagy inhibitors would be expected to prevent the onset of drug resistance. We also discuss why anti-angiogenic therapy has been found to promote tumor recurrence, progression and metastasis. More specifically, anti-angiogenic therapy would induce autophagy in the tumor stroma via the induction of stromal hypoxia, thereby converting a non-aggressive tumor type to a “lethal” aggressive tumor phenotype. Thus, uncoupling the metabolic parasitic relationship between cancer cells and an autophagic tumor stroma may hold great promise for anti-cancer therapy. Finally, we believe that autophagy in the tumor stroma is the local microscopic counterpart of systemic wasting (cancer-associated cachexia), which is associated with advanced and metastatic cancers. Cachexia in cancer patients is not due to decreased energy intake, but instead involves an increased basal metabolic rate and increased energy expenditures, resulting in a negative energy balance. Importantly, when tumors were surgically excised, this increased metabolic rate returned to normal levels. This view of cachexia, resulting in energy transfer to the tumor, is consistent with our hypothesis. So, cancer-associated cachexia may start locally as stromal autophagy and then spread systemically. As such, stromal autophagy may be the requisite precursor of systemic cancer-associated cachexia.Key words: caveolin-1, autophagy, cancer associated fibroblasts, hypoxia, mitophagy, oxidative stress, DNA damage, genomic instability, tumor stroma, wasting (cancer cachexia), Warburg effect     

Mitochondrial metabolism in cancer metastasis: Visualizing tumor cell mitochondria and the “reverse Warburg effect” in positive lymph node tissue

We have recently proposed a new two-compartment model for understanding the Warburg effect in tumor metabolism. In this model, glycolytic stromal cells produce mitochondrial fuels (L-lactate and ketone bodies) that are then transferred to oxidative epithelial cancer cells, driving OXPHOS and mitochondrial metabolism. Thus, stromal catabolism fuels anabolic tumor growth via energy transfer. We have termed this new cancer paradigm the “reverse Warburg effect,” because stromal cells undergo aerobic glycolysis, rather than tumor cells. To assess whether this mechanism also applies during cancer cell metastasis, we analyzed the bioenergetic status of breast cancer lymph node metastases, by employing a series of metabolic protein markers. For this purpose, we used MCT4 to identify glycolytic cells. Similarly, we used TOMM20 and COX staining as markers of mitochondrial mass and OXPHOS activity, respectively. Consistent with the “reverse Warburg effect,” our results indicate that metastatic breast cancer cells amplify oxidative mitochondrial metabolism (OXPHOS) and that adjacent stromal cells are glycolytic and lack detectable mitochondria. Glycolytic stromal cells included cancer-associated fibroblasts, adipocytes and inflammatory cells. Double labeling experiments with glycolytic (MCT4) and oxidative (TOMM20 or COX) markers directly shows that at least two different metabolic compartments co-exist, side-by-side, within primary tumors and their metastases. Since cancer-associated immune cells appeared glycolytic, this observation may also explain how inflammation literally “fuels” tumor progression and metastatic dissemination, by “feeding” mitochondrial metabolism in cancer cells. Finally, MCT4(+) and TOMM20(-) “glycolytic” cancer cells were rarely observed, indicating that the conventional “Warburg effect” does not frequently occur in cancer-positive lymph node metastases.Key words: caveolin-1, oxidative stress, MCT4, metabolic coupling, tumor stroma, SLC16A3, monocarboxylic acid transporter, two-compartment tumor metabolism, metastasis, TOMM20, complex IV, OXPHOS, mitochondria, inflammation     

Evidence for a stromal-epithelial “lactate shuttle” in human tumors: MCT4 is a marker of oxidative stress in cancer-associated fibroblasts

Recently, we proposed a new mechanism for understanding the Warburg effect in cancer metabolism. In this new paradigm, cancer-associated fibroblasts undergo aerobic glycolysis, and extrude lactate to “feed” adjacent cancer cells, which then drives mitochondrial biogenesis and oxidative mitochondrial metabolism in cancer cells. Thus, there is vectorial transport of energy-rich substrates from the fibroblastic tumor stroma to anabolic cancer cells. A prediction of this hypothesis is that cancer-associated fibroblasts should express MCT4, a mono-carboxylate transporter that has been implicated in lactate efflux from glycolytic muscle fibers and astrocytes in the brain. To address this issue, we co-cultured MCF7 breast cancer cells with normal fibroblasts. Interestingly, our results directly show that breast cancer cells specifically induce the expression of MCT4 in cancer-associated fibroblasts; MCF7 cells alone and fibroblasts alone, both failed to express MCT4. We also show that the expression of MCT4 in cancer-associated fibroblasts is due to oxidative stress, and can be prevented by pre-treatment with the anti-oxidant N-acetyl-cysteine. In contrast to our results with MCT4, we see that MCT1, a transporter involved in lactate uptake, is specifically upregulated in MCF7 breast cancer cells when co-cultured with fibroblasts. Virtually identical results were also obtained with primary human breast cancer samples. In human breast cancers, MCT4 selectively labels the tumor stroma, e.g., the cancer-associated fibroblast compartment. Conversely, MCT1 was selectively expressed in the epithelial cancer cells within the same tumors. Functionally, we show that overexpression of MCT4 in fibroblasts protects both MCF7 cancer cells and fibroblasts against cell death, under co-culture conditions. Thus, we provide the first evidence for the existence of a stromal-epithelial lactate shuttle in human tumors, analogous to the lactate shuttles that are essential for the normal physiological function of muscle tissue and brain. These data are consistent with the “reverse Warburg effect,” which states that cancer-associated fibroblasts undergo aerobic glycolysis, thereby producing lactate, which is utilized as a metabolic substrate by adjacent cancer cells. In this model, “energy transfer” or “metabolic-coupling” between the tumor stroma and epithelial cancer cells “fuels” tumor growth and metastasis, via oxidative mitochondrial metabolism in anabolic cancer cells. Most importantly, our current findings provide a new rationale and novel strategy for anti-cancer therapies, by employing MCT inhibitors.Key words: caveolin-1, oxidative stress, pseudohypoxia, lactate shuttle, MCT1, MCT4, metabolic coupling, tumor stroma, predictive biomarker, SLC16A1, SLC16A3, monocarboxylic acid transporter     

Emergence,Development, and Maturity of the Gonad of Two Species of Chitons “Sea Cockroach” (Mollusca: Polyplacophora) through the Early Life Stages

This study describes and recognises, using histological and microscopical examinations on a morphometrical basis, several gonad traits through the early life stages of Chiton articulatus and C. albolineatus. Gonadal ontogenesis, gonad development stages, sexual differentiation, onset of the first sexual maturity, and growth sequences or “early life stages” were determined. In addition, allometry between lengths and body weight pooled for both sexes per each chiton were calculated using equation Y = aX^b. A total of 125 chitons (4≤TL≤40 mm, in total length “TL”) were used. All allometric relations showed a strong positive correlation (r), close to 1, with b-values above three, indicating an isometric growth. Gonadal ontogenesis and gonad development stages were categorised into three periods (“Pw” without gonad, “Pe” gonad emergence, and “Pf” gonadal sac formed) and four stages (“S0” gametocytogenesis, “S1” gametogenesis, “S2” mature, and “S3” spawning), respectively. Compound digital images were attained for each process. Periods and stages are overlapped among them and between species, with the following overall confidence intervals in TL: Pw 6.13–14.32 mm, Pe 10.32–16.93 mm, Pf 12.99–25.01 mm, S0 16.08–24.34 mm (females) and 19.51–26.60 mm (males), S1 27.15–35.63 mm (females) and 23.45–32.27 mm (males), S2 24.48–40.24 mm (females) and 25.45–32.87 mm (males). Sexual differentiation (in S0) of both chitons occurs first as a female then as a male; although, males reach the onset of the first sexual maturity earlier than females, thus for C. articulatus males at 17 mm and females at 32 mm, and for C. albolineatus males at 23.5 mm and females at 28 mm, all in TL. Four early life stages (i.e., subjuvenile, juvenile, subadult, and adult) are described and proposed to distinguish growth sequences. Our results may be useful to diverse disciplines, from developmental biology to fisheries management.     

Characterization of the Denitrification-Associated Phosphorus Uptake Properties of “Candidatus Accumulibacter phosphatis” Clades in Sludge Subjected to Enhanced Biological Phosphorus Removal

To characterize the denitrifying phosphorus (P) uptake properties of “Candidatus Accumulibacter phosphatis,” a sequencing batch reactor (SBR) was operated with acetate. The SBR operation was gradually acclimated from anaerobic-oxic (AO) to anaerobic-anoxic-oxic (A2O) conditions by stepwise increases of nitrate concentration and the anoxic time. The communities of “Ca. Accumulibacter” and associated bacteria at the initial (AO) and final (A2O) stages were compared using 16S rRNA and polyphosphate kinase genes and using fluorescence in situ hybridization (FISH). The acclimation process led to a clear shift in the relative abundances of recognized “Ca. Accumulibacter” subpopulations from clades IIA > IA > IIF to clades IIC > IA > IIF, as well as to increases in the abundance of other associated bacteria (Dechloromonas [from 1.2% to 19.2%] and “Candidatus Competibacter phosphatis” [from 16.4% to 20.0%]), while the overall “Ca. Accumulibacter” abundance decreased (from 55.1% to 29.2%). A series of batch experiments combined with FISH/microautoradiography (MAR) analyses was performed to characterize the denitrifying P uptake properties of the “Ca. Accumulibacter” clades. In FISH/MAR experiments using slightly diluted sludge (∼0.5 g/liter), all “Ca. Accumulibacter” clades successfully took up phosphorus in the presence of nitrate. However, the “Ca. Accumulibacter” clades showed no P uptake in the presence of nitrate when the sludge was highly diluted (∼0.005 g/liter); under these conditions, reduction of nitrate to nitrite did not occur, whereas P uptake by “Ca. Accumulibacter” clades occurred when nitrite was added. These results suggest that the “Ca. Accumulibacter” cells lack nitrate reduction capabilities and that P uptake by “Ca. Accumulibacter” is dependent upon nitrite generated by associated nitrate-reducing bacteria such as Dechloromonas and “Ca. Competibacter.”     

Immunodominant HIV-Specific CD8+ T-Cell Responses Are Common to Blood and Gastrointestinal Mucosa,and Gag-Specific Responses Dominate in Rectal Mucosa of HIV Controllers

Previous studies have suggested that polyfunctional mucosal CD8⁺ T-cell responses may be a correlate of protection in HIV controllers. Mucosal T-cell breadth and/or specificity may also contribute to defining protective responses. In this study, rectal CD8⁺ T-cell responses to HIV Gag, Env, and Nef were mapped at the peptide level in four subject groups: elite controllers (n = 16; viral load [VL], <75 copies/ml), viremic controllers (n = 14; VL, 75 to 2,000 copies/ml), noncontrollers (n = 14; VL, >10,000 copies/ml), and antiretroviral-drug-treated subjects (n = 8; VL, <75 copies/ml). In all subject groups, immunodominant CD8⁺ T-cell responses were generally shared by blood and mucosa, although there were exceptions. In HIV controllers, responses to HLA-B27- and HLA-B57-restricted epitopes were common to both tissues, and their magnitude (in spot-forming cells [SFC] per million) was significantly greater than those of responses restricted by other alleles. Furthermore, peptides recognized by T cells in both blood and rectal mucosa, termed “concordant,” elicited higher median numbers of SFC than discordant responses. In magnitude as well as breadth, HIV Gag-specific responses, particularly those targeting p24 and p7, dominated in controllers. Responses in noncontrollers were more evenly distributed among epitopes in Gag, Env, and Nef. Viremic controllers showed significantly broader mucosal Gag-specific responses than other groups. Taken together, these findings demonstrate that (i) Gag-specific responses dominate in mucosal tissues of HIV controllers; (ii) there is extensive overlap between CD8⁺ T cells in blood and mucosal tissues, with responses to immunodominant epitopes generally shared by both sites; and (iii) mucosal T-cell response breadth alone cannot account for immune control.Despite more than two decades of intensive research, the immunologic correlates of protection from human immunodeficiency virus (HIV) infection and disease progression remain incompletely understood. To date, the majority of studies of HIV-specific T-cell responses have focused on the measurement of such responses in peripheral blood lymphocytes. Nevertheless, the majority of the body''s lymphocytes are housed in mucosal tissues, notably the gastrointestinal (GI) tract (18, 33, 40). The gastrointestinal mucosa also serves as a major target of HIV infection and CD4⁺ T-cell depletion (7, 25, 36), as well as an important site of transmission (18, 33, 40). Antigen-experienced T cells may preferentially traffic to tissue sites of infection (50), where they may also expand in an antigen-driven manner. Because of the unique role of the gastrointestinal mucosa in HIV pathogenesis, detailed studies of HIV-specific immune responses in this compartment may contribute important insights to our understanding of the disease process.An important question is the degree to which T-cell responses in mucosal tissues are “compartmentalized” and distinct in specificity and/or clonality from those found elsewhere in the body, including in peripheral blood. Because of the technical challenges associated with obtaining large numbers of viable lymphocytes from mucosal biopsy specimen tissue, comprehensive mapping of the fine specificity of mucosal HIV-specific T-cell responses has been difficult. Relying on a polyclonal expansion approach, Ibarrondo and colleagues successfully mapped HIV-specific CD8⁺ T-cell responses in blood and rectal mucosa of chronically infected persons to the level of peptide pools but not to individual epitopes (29). Their studies revealed a similar pattern of responses, and nearly identical immunodominance hierarchies, in the two tissue sites.We have focused our recent studies of mucosal immunity on a group of individuals who control HIV infection in the absence of antiretroviral therapy. These are often called “long-term nonprogressors” (LTNP) (14), referring to their ability to maintain normal CD4⁺ T-cell counts for more than 10 years without medication. LTNP are believed to account for 5 to 15% of the HIV-infected population. Several recent studies have used the term “HIV controllers,” defined as those who maintain undetectable plasma HIV RNA levels (“elite controllers”) and those who have persistently detectable but low plasma HIV RNA levels (“viremic controllers”). Elite controllers represent less than 1% of the HIV-infected population (14). In contrast, individuals with viral loads of >10,000 copies/ml in the absence of therapy are termed “noncontrollers.” Recently, we found that “polyfunctional” HIV-specific T cells, producing multiple antiviral factors, were significantly more abundant in gastrointestinal mucosa of HIV controllers than in those of noncontrollers or subjects on highly active antiretroviral therapy (HAART) (20). Furthermore, in many cases these strong, polyfunctional mucosal T-cell responses were not mirrored in peripheral blood, suggesting that HIV-specific T cells either preferentially traffic to or undergo expansion within mucosal tissues.Because of these findings, we undertook a follow-up study to determine the breadth and fine specificity, to the peptide level, of mucosal CD8⁺ T-cell responses to HIV Gag, Env, and Nef among HIV controllers, noncontrollers, and individuals on HAART. We hypothesized that controllers might harbor an unusually broad repertoire of HIV-specific CD8⁺ T cells in mucosal tissues. We found a similar response breadth in mucosal tissues of all three subject groups, arguing against a critical role for mucosal T-cell response breadth in determining the extent of HIV control. In contrast, we found that high-magnitude mucosal responses directed at well-conserved regions in Gag were a strong and consistent correlate of control. Finally, concordant responses, defined as those common to blood and mucosa, were generally stronger than discordant responses, underscoring the observation that T cells responding to immunodominant epitopes are broadly distributed throughout the body in both controllers and noncontrollers.     

Fibroblast Growth Factor Receptors-1 and -3 Play Distinct Roles in the Regulation of Bladder Cancer Growth and Metastasis: Implications for Therapeutic Targeting

Fibroblast growth factor receptors (FGFRs) are activated by mutation and overexpressed in bladder cancers (BCs), and FGFR inhibitors are currently being evaluated in clinical trials in BC patients. However, BC cells display marked heterogeneity in their responses to FGFR inhibitors, and the biological mechanisms underlying this heterogeneity are not well defined. Here we used a novel inhibitor of FGFRs 1–3 and RNAi to determine the effects of inhibiting FGFR1 or FGFR3 in a panel of human BC cell lines. We observed that FGFR1 was expressed in BC cells that also expressed the “mesenchymal” markers ZEB1 and vimentin, whereas FGFR3 expression was restricted to the E-cadherin- and p63-positive “epithelial” subset. Sensitivity to the growth-inhibitory effects of BGJ-398 was also restricted to the “epithelial” BC cells and it correlated directly with FGFR3 mRNA levels but not with the presence of activating FGFR3 mutations. In contrast, BGJ-398 did not strongly inhibit proliferation but did block invasion in the “mesenchymal” BC cells in vitro. Similarly, BGJ-398 did not inhibit primary tumor growth but blocked the production of circulating tumor cells (CTCs) and the formation of lymph node and distant metastases in mice bearing orthotopically implanted “mesenchymal” UM-UC3 cells. Together, our data demonstrate that FGFR1 and FGFR3 have largely non-overlapping roles in regulating invasion/metastasis and proliferation in distinct “mesenchymal” and “epithelial” subsets of human BC cells. The results suggest that the tumor EMT phenotype will be an important determinant of the biological effects of FGFR inhibitors in patients.     

Human Blood and Mucosal Regulatory T Cells Express Activation Markers and Inhibitory Receptors in Inflammatory Bowel Disease

Background

FOXP3⁺ regulatory T cells (Tregs) are critical for preventing intestinal inflammation. However, FOXP3⁺ T cells are paradoxically increased in the intestines of patients with the inflammatory bowel disease (IBD) ulcerative colitis (UC) or Crohn’s disease (CD). We determined whether these FOXP3⁺ cells in IBD patients share or lack the phenotype of such cells from patients without IBD.

Methods

We quantified and characterized FOXP3⁺ Treg populations, as well as FOXP3^- CD4⁺ T cells, in the lamina propria lymphocytes (LPL) of intestine surgically resected from patients with and without IBD, and in the blood of controls or Crohn’s patients with or without disease activity.

Results

In all samples, a similar fraction of FOXP3⁺ cells expressed the “natural” Treg (nTreg) marker Helios, suggesting that, in IBD, these cells are not entirely “induced” Tregs (iTregs) derived from activated effector T cells. Helios⁺ and Helios^- FOXP3⁺ T cells demonstrated similar expression of maturation markers, activation markers, and inhibitory molecules between IBD patients and controls, while FOXP3^- cells paradoxically expressed more of the inhibitory receptors CD39, CTLA4, and PD-1 in inflamed mucosa. Greater expression of activation markers was also seen in both Helios⁺ and Helios^- Tregs, relative to FOXP3^- cells, in both IBD patients and controls, indicating that Tregs are effectively activated by antigen in IBD.

Conclusions

Extensive immunophenotyping revealed that Helios⁺ and Helios^- mucosal Tregs exist at a similar frequency, and have a similar expression of inhibitory molecules and activation markers in patients with IBD as in healthy controls.     

The Metabolism of the Volatile Amines: VII. The Clinical Significance of Two Components of the Blood Ammonia Level

Blood ammonia levels consist of two components: ammonia present in blood at the time of shedding, termed “free” ammonia, and ammonia produced by the deamidating action of the alkali reagents. Blood of healthy people contained little or no “free” ammonia while blood of patients with chronic liver disease occasionally showed levels up to 1.2 μg./ml. Patients with hepatic encephalopathy had significantly elevated levels which usually fell to zero following therapy. Levels of “free” ammonia above 0.6 μg./ml. were diagnostic of hepatic encephalopathy in patients suffering from unexplained neurological disorders.The rate of formation of ammonia by the alkali reagents was increased in patients with hepatic necrosis and was depressed in those with chronic hepatitis. The ammonia appeared to arise from the deamidation of glutamine and asparagine, present in blood in both the free and peptide forms.