Allergic disease in 8-year-old children is preceded by delayed B cell maturation.

BACKGROUND: We previously reported that exposure to a farming environment is allergy-protective, while high proportions of neonatal immature/naïve CD5+ B cells and putative regulatory T cells (Tregs) are risk factors for development of allergic disease and sensitization up to 3 years of age. OBJECTIVE: To examine if B and T cell maturation are associated with allergic disease and farming environment over the first 8 years in life. METHODS: In the prospective FARMFLORA study, including both farming and non-farming families, 48 of 65 children took part in the 8-year follow-up study. Various B and T cell maturation variables were examined in blood samples obtained at several occasions from birth to 8 years of age and related to doctors' diagnosed allergic disease and sensitization, and to farming environment. RESULTS: We found that the incidence of allergic disease was lower among farmers' compared to non-farmers' children during the 8-year follow-up period, and that farmers' children had higher proportions of memory B cells at 8 years of age. Moreover, a high proportion of neonatal CD5+ B cells was a risk factor for and may predict development of allergic disease at 8 years of age. A high proportion of Tregs was not protective against development of these conditions. CONCLUSION AND CLINICAL RELEVANCE: High proportions of neonatal naïve B cells remained as a risk factor for allergic disease in school-aged children. Thus, the accelerated B cell maturation observed among farmers' children may be crucial for the allergy-protective effect of a farming environment.

Enveloped virus but not bacteria block IL-13 responses in human cord blood T cells in vitro.

Infections that occur early in life may have a beneficial effect on the immune system and thereby reduce the risk of allergen sensitization and/or allergic disease. It is not yet clear to what extent specific virus and/or bacteria can mediate this effect. The purpose of this study was to assess the role of virus and bacteria in CD4(+) T cell-derived cytokine production in newborns. We compared the effects of five bacteria (Staphlococcus aureus, Escherichia coli, Clostridium difficile, Lactobacillus rhamnosus and Bifidobacterium bifidus) and seven virus (adenovirus, coronavirus, cytomegalovirus, herpes simplex virus, influenza virus, morbillivirus and poliovirus) on the Th1/Th2 cytokine production in mixed lymphocyte reactions using CD4(+) T cells from cord blood cocultured with allogenic myeloid or plasmacytoid dendritic cells. When comparing the baseline cytokine production prior to microbial stimulation, we observed that cord plasmacytoid DC were stronger inducers of Th2 cytokines (IL-5 and IL-13) compared with cord myeloid DC and to adult DC. When adding microbes to these cultures, bacteria and virus differed in two major respects; Firstly, all enveloped viruses, but none of the bacteria, blocked Th2 (IL-13) production by cord CD4(+) cells. Secondly, all Gram-positive bacteria, but none of the virus, induced IL-12p40 responses, but the IL-12p40 responses did not affect Th1 cytokine production (IFN-γ). Instead, Th1 responses were correlated with the capacity to induce IFN-α secretion, which in cord cells were induced by S. aureus and influenza virus alone. These data imply that enveloped virus can deviate Th2 responses in human cord T cells.

Infection of infants with human herpesvirus type 6 may be associated with reduced allergic sensitization and T-helper type 2 development.

BACKGROUND: Epidemiological studies point to an inverse relationship between microbial exposure and the prevalence of allergic diseases. The underlying mechanism for this observation remains largely unknown, as well as the nature of the microbes involved. OBJECTIVE: To investigate the effects of early infection with human herpesviruses (HHVs) on IgE formation and T-helper type 2 (Th2) development in infants. METHODS: Serum was collected from children aged 18 months and assessed for IgE to common allergens and IgG to five common herpesviruses. Cord blood plasmacytoid dendritic cells (pDC) were exposed to HHV type 6 in vitro and mixed with allogeneic cord blood CD4(+) T cells. Cytokine levels were determined by ELISA and by flow cytometry. RESULTS: We found that children seropositive at 18 months of age to HHV type 6 were significantly less often IgE sensitized than seronegative children [odds ratio (OR): 0.08, 95% confidence interval (CI): 0.009-0.68]. HHV type 6 also decreased the production of the Th2-associated cytokines IL-5 and IL-13 by CD4(+) T cells when co-cultured with allogeneic cord blood pDC. This was associated with an increased production of IFN-alpha by pDC exposed to HHV type 6. CONCLUSION: These data indicate that an early childhood infection with HHV type 6 could down-regulate Th2 responses and reduce IgE formation to common allergens in a young child.

High circulating immunoglobulin A levels in infants are associated with intestinal toxigenic Staphylococcus aureus and a lower frequency of eczema.

BACKGROUND: Intestinal bacteria trigger IgA production and delayed maturation of mucosal IgA response is linked to allergy development. OBJECTIVE: Our aim was to investigate if plasma levels of IgA or APRIL (a proliferation inducing ligand), an important factor for IgA class switch recombination, in infancy correlates with intestinal colonization by any specific bacteria or yeast. We also examined if plasma IgA or APRIL levels are related to sensitization and the development of eczema. METHODS: IgA was quantified in plasma obtained from infants at birth and at 4 and 18 months of age and APRIL was measured at 4 months of age. Colonization by major bacterial groups and yeast was followed in the first 8 weeks of life by quantitative culture of stool samples. A clinical evaluation regarding the presence of allergen-specific IgE or eczema and eosinophil counts in blood was performed at 18 months of age. RESULTS: In multiple linear regression analysis, only colonization by Staphylococcus aureus strains producing toxins with superantigen function (SEA-D or TSST-1) made an independent contribution to plasma IgA levels at 4 months of age. Further, increased levels of APRIL in plasma at 4 months were negatively associated with sensitization while IgA plasma levels were inversely correlated to eczema development and blood eosinophil counts at 18 months of age. CONCLUSION: Early intestinal colonization by toxigenic S. aureus strains seems to promote systemic IgA responses. Furthermore, high levels of APRIL and IgA in the circulation at 4 months of age seem to correlate negatively with allergy development.

Characterization of T-cell reactive epitopes in glycoprotein G of herpes simplex virus type 2 using synthetic peptides.

We have previously shown that the CD4+ T-cell response to herpes simplex virus type 2 glycoprotein G-2 is type-specific and can thus be used to evaluate herpes simplex virus type 2-specific T-cell responses in individuals with a concomitant herpes simplex virus type 1 infection. In this study we have followed the glycoprotein G-2-specific T-cell responses over time, and also tried to identify T-cell epitopes in the membrane bound portion and the secreted portion of glycoprotein G-2 using synthetic peptides spanning the whole amino acid sequence of glycoprotein G-2. We found that the magnitude of the glycoprotein G-2-specific response varied considerably in infected individuals over time, even though all patients responded to at least one of the two glycoproteins at all time-points examined. We could also document strong T-cell responses to synthetic peptides from the secreted glycoprotein G-2 but only low responses to synthetic peptides corresponding to sequences from the heavily glycosylated membrane-bound glycoprotein G-2. We were able to map an immunogenic region (amino acid 31-125) within the secreted glycoprotein G-2. This region of the glycoprotein induced proliferative responses in 47% of the herpes simplex virus type 2-infected individuals. However, we were not able to identify any universal T-cell epitope.

Cholera toxin B subunit as a carrier molecule promotes antigen presentation and increases CD40 and CD86 expression on antigen-presenting cells.

Cholera toxin B subunit (CTB) is an efficient mucosal carrier molecule for the generation of mucosal antibody responses and/or induction of systemic T-cell tolerance to linked antigens. CTB binds with high affinity to GM1 ganglioside cell surface receptors. In this study, we evaluated how conjugation of a peptide or protein antigen to CTB by chemical coupling or genetic fusion influences the T-cell-activating capacity of different antigen-presenting cell (APC) subsets. Using an in vitro system in which antigen-pulsed APCs were incubated with antigen-specific, T-cell receptor-transgenic T cells, we found that the dose of antigen required for T-cell activation could be decreased >10,000-fold using CTB-conjugated compared to free antigen. In contrast, no beneficial effects were observed when CTB was simply admixed with antigen. CTB conjugation enhanced the antigen-presenting capacity not only of dendritic cells and B cells but also of macrophages, which expressed low levels of cell surface major histocompatibility complex (MHC) class II and were normally poor activators of naive T cells. Enhanced antigen-presenting activity by CTB-linked antigen resulted in both increased T-cell proliferation and increased interleukin-12 and gamma interferon secretion and was associated with up-regulation of CD40 and CD86 on the APC surface. These results imply that conjugation to CTB dramatically lowers the threshold concentration of antigen required for immune cell activation and also permits low-MHC II-expressing APCs to prime for a specific immune response.

Vaccination with Bordetella pertussis-pulsed autologous or heterologous dendritic cells induces a mucosal antibody response in vivo and protects against infection.

This study demonstrates for the first time that vaccination with either autologous or heterologous dendritic cells (DC) pulsed with specific antigen induces protective immune responses against noninvasive bacteria, namely Bordetella pertussis. The DC-mediated protection is associated with strong B. pertussis-specific immunoglobulin G (IgG) and IgA responses in the lung.

Local and systemic immune responses to rectal administration of recombinant cholera toxin B subunit in humans.

The induction of immune responses to rectally administered recombinant cholera toxin B subunit (CTB) in humans was studied. Three immunizations induced high levels of CTB-specific antibody-secreting cells, particular of the immunoglobulin A isotype, in both rectum and peripheral blood. Antitoxin antibody responses in rectal secretions and serum were also found.

Dendritic cell vaccination protects mice against lethality caused by genital herpes simplex virus type 2 infection.

We have evaluated the ability of antigen pulsed bone-marrow derived dendritic cells (bmDC), to induce protective immunity against a genital tract infection with herpes simplex virus type 2 (HSV-2) in mice. Intravenous but not vaginal administrations of bmDC pulsed in vitro with UV-inactivated HSV-2, or with purified HSV-2 envelope glycoproteins gave rise to complete protection against disease, as well as death caused by genital herpes infection. Protection was dependent on the antigens being presented by the bmDC as neither the antigens alone, nor the mock-pulsed bmDC prevented disease. Immunity was associated with HSV-2 specific IFN-gamma and antibody production, and was shown to be dependent on CD4(+) cells secreting IFN-gamma. Thus, ex vivo antigen-pulsed bmDC represents a powerful tool for the study of protective immunity to genital herpes infection, and for the identification of protective antigens. These findings might also have an impact on the design of vaccines against other sexually transmitted viral diseases.

Rat arterial smooth muscle devoid of ryanodine receptor function: effects on cellular Ca(2+) handling.

The roles of intracellular Ca(2+) stores and ryanodine (Ry) receptors for vascular Ca(2+) homeostasis and viability were investigated in rat tail arterial segments kept in organ culture with Ry (10 - 100 microM) for up to 4 days. Acute exposure to Ry or the non-deactivating ryanodine analogue C(10)-O(eq) glycyl ryanodine (10 microM) eliminated Ca(2+) release responses to caffeine (20 mM) and noradrenaline (NA, 10 microM), whereas responses to NA, but not caffeine, gradually returned to normal within 4 days of exposure to RY: Ry receptor protein was detected on Western blots in arteries cultured either with or without RY: Brief Ca(2+) release events (sparks) were absent after culture with Ry, whereas Ca(2+) waves still occurred. The propagation velocity of waves was equal ( approximately 19 microm s(-1)) in tissue cultured either with or without RY: Inhibition of Ca(2+) accumulation into the sarcoplasmic reticulum (SR) by culture with caffeine (5 mM), cyclopiazonic acid or thapsigargin (both 10 microM) decreased contractility due to Ca(2+)-induced cell damage. In contrast, culture with Ry did not affect contractility. Removal of Ca(2+) from the cytosol following a Ca(2+) load was retarded after Ry culture. Thapsigargin reduced the rate of Ca(2+) removal in control cultured rings, but had no effect after Ry culture. It is concluded that intracellular Ca(2+) stores recover during chronic Ry treatment, while Ry receptors remain non-functional. Ry receptor activity is required for Ca(2+) sparks and for SR-dependent recovery from a Ca(2+) load, but not for Ca(2+) waves or basal Ca(2+) homeostasis.

Stretch-dependent modulation of contractility and growth in smooth muscle of rat portal vein.

Increased intraluminal pressure of the rat portal vein in vivo causes hypertrophy and altered contractility in 1 to 7 days. We have used organ cultures to investigate mechanisms involved in this adaptation to mechanical load. Strips of rat portal vein were cultured for 3 days, either undistended or loaded by a weight. Length-force relations were shifted toward longer length in stretched cultured veins compared with freshly dissected veins, whereas the length-force relations of unstretched cultured veins were shifted in the opposite direction. This occurred after culture either with or without 10% FCS to promote growth. The wet weight of loaded veins increased by 56% in the presence of FCS, whereas that of undistended control veins increased by 24%. No weight increase was seen in serum-free culture. The dry/wet weight ratio decreased during culture with FCS but was not affected by stretch. Electron microscopy revealed increased cell cross-sectional area in stretched relative to unstretched veins, and protein contents were greater, as were [(3)H]thymidine and [(3)H]leucine incorporation rates. Growth responses were associated with the activation of stretch-sensitive extracellular signal-regulated kinases 1 and 2 and were inhibited by herbimycin A and PD 98059, inhibitors of extracellular signal-regulated kinases 1 and 2. The results demonstrate that by culture of whole vascular tissue, smooth muscle cells are maintained in the contractile phenotype and respond to stretch with a physiological adaptation involving hypertrophy/hyperplasia and remodeling of the contractile system, similar to that in vivo. Mechanical stimulation and growth factors are both required for functionally significant growth.

Differential effect of cholera toxin on CD45RA+ and CD45RO+ T cells: specific inhibition of cytokine production but not proliferation of human naive T cells.

We have studied how cholera toxin (CT) and its non-toxic cell-binding B-subunit (CTB) affect the activation of pure human T cells in an anti-CD3-driven system. CT, as opposed to CTB, strongly suppressed the proliferative responses as well as cytokine production in CD4+ and CD8+ T cells. CT however, had a differential effect on naive and activated/memory T cell subsets. Costimulation through exogenous IL-2 or through CD28 cross-linking rescued the proliferation of CT-treated naive CD45RA+ T cells, but not of activated/memory CD45RO+ cells. IL-2 production and IL-2 receptor expression were markedly reduced by CT in all T cell fractions, i.e. also in CD45RA+ cells which had maintained proliferative responses. However, the proliferative responses of CT-treated CD45RA+ T cells were IL-2-dependent, as shown by blocking experiments using anti-IL-2 antibodies. These results indicate (i) that CTB has no cytostatic effect on human T cells, (ii) that CT affects proliferation and cytokine production by two different signal pathways, and (iii) that CT might interact with a signal pathway generated through or influenced by CD45.

Prolonged oral treatment with low doses of allergen conjugated to cholera toxin B subunit suppresses immunoglobulin E antibody responses in sensitized mice.

BACKGROUND: Oral tolerance is a long recognized method for inducing systemic immunological tolerance. However, large doses of antigen and frequent administrations are often required. By linking the antigen to the nontoxic mucosa-binding B subunit of cholera toxin (CTB), the required amount can be dramatically reduced. We have previously shown that mucosal administration of small amounts of antigens coupled to CTB can suppress peripheral Th1 cell-reactivity and associated inflammatory immunopathology in both naive and systemically-immunized animals. Induction of oral tolerance by repeated feeding of relatively small doses of antigen has, in some cases been shown to involve the generation of regulatory Th2-like CD4+ T cells, and hence could promote rather than suppress type I immunoglobulin (Ig) E-mediated allergic responses. OBJECTIVES: We examined whether oral prophylactic or therapeutic administration of a model allergen coupled to CTB would modulate allergen-specific IgE responses in high IgE responder Balb/c mice. METHODS: Ovalbumin (OVA) was used as a model allergen. Mice were treated perorally with free or CTB-coupled OVA before or after systemic priming with alum-adsorbed OVA. Allergen-specific IgE levels in serum were measured with the passive cutaneous anaphylaxis test at various time-points. RESULTS: Oral administration of a single low dose of CTB-linked OVA, prior to systemic sensitization and challenge with OVA, suppressed allergen-specific serum IgE antibody responses. Treatment with comparable doses of free OVA was much less effective. Most importantly, oral treatment with CTB-OVA conjugate could also suppress an already initiated IgE antibody response, but to achieve such a 'therapeutic effect', administration of multiple low doses of conjugate over a long time was required. Oral treatment with CTB-OVA conjugate could also effectively suppress antigen-specific Th1-mediated delayed-type hypersensitivity. Thus treatment with a CTB-conjugated model allergen can affect a broad range of T-cell-driven immune responses, even in antigen-experienced animals. CONCLUSION: These results may impact on the development of therapeutic vaccines against type I allergies.

Genital antibody responses in mice after intranasal infection with an attenuated candidate vector strain of Bordetella pertussis.

Intranasal administration of live attenuated Bordetella pertussis, from which the pertussis toxin gene has been deleted, has previously been shown to give rise to high levels of serum immunoglobulin G (IgG) antibodies against both the protective antigen filamentous hemagglutinin (FHA) and heterologous antigens genetically fused to FHA. Here, we extend these results by demonstrating that anti-FHA IgA and IgG antibodies are also produced in the genital tract of mice, both in the vagina and in the uterus, after a single intranasal administration of B. pertussis. By comparing the immune responses induced after infection with wild-type virulent B. pertussis with that induced by infection with an attenuated pertussis toxin-deficient strain, we conclude that pertussis toxin produced by the virulent bacteria does not modify antibody production to FHA in the genital tract of B. pertussis-infected mice. The intranasal infection with either the attenuated or the virulent B. pertussis strain also led to the development of immunologic memory that could be efficiently boosted with purified FHA administered either intranasally or intravaginally to give rise to a significant increase in the levels of specific IgA and IgG produced locally in the genital tract, as well as of specific antibodies in the serum. These observations suggest that attenuated B. pertussis could be a promising vector for intranasal administration to induce antibody responses against antigens from sexually transmitted pathogens fused to FHA.

Anatomic segmentation of the intestinal immune response in nonhuman primates: differential distribution of B cells after oral and rectal immunizations to sites defined by their source of vascularization.

We show that the distribution of specific antibodies and antibody-secreting cells in the intestine after oral and rectal immunizations corresponds to the vascularization and lymph drainage patterns of the gut. Oral immunizations induce antibody responses along the parts of the intestine connected to the superior mesenteric vessels and lymph ducts, whereas rectal immunizations induce antibody responses along the parts of the intestine associated with the inferior mesenteric vessels and ducts.

Long-term effects of Ca(2+) on structure and contractility of vascular smooth muscle.

Culture of dispersed smooth muscle cells is known to cause rapid modulation from the contractile to the synthetic cellular phenotype. However, organ culture of smooth muscle tissue, with maintained extracellular matrix and cell-cell contacts, may facilitate maintenance of the contractile phenotype. To test the influence of culture conditions, structural, functional, and biochemical properties of rat tail arterial rings were investigated after culture. Rings were cultured for 4 days in the absence and presence of 10% FCS and then mounted for physiological experiments. Intracellular Ca(2+) concentration ([Ca(2+)](i)) after stimulation with norepinephrine was similar in rings cultured with and without FCS, whereas force development after FCS was decreased by >50%. The difference persisted after permeabilization with beta-escin. These effects were associated with the presence of vasoconstrictors in FCS and were dissociated from its growth-stimulatory action. FCS treatment increased lactate production but did not affect ATP, ADP, or AMP contents. The contents of actin and myosin were decreased by culture but similar for all culture conditions. There was no effect of FCS on calponin contents or myosin SM1/SM2 isoform composition, nor was there any appearance of nonmuscle myosin. FCS-stimulated rings showed evidence of cell degeneration not found after culture without FCS or with FCS + verapamil (1 microM) to lower [Ca(2+)](i). The decreased force-generating ability after culture with FCS is thus associated with increased [Ca(2+)](i) during culture and not primarily caused by growth-associated modulation of cells from the contractile to the synthetic phenotype.

Combined immunomagnetic cell sorting and ELISPOT assay for the phenotypic characterization of specific antibody-forming cells.

A combination of immunomagnetic cell sorting and ELISPOT techniques has been evaluated to permit enrichment and characterization of antibody-secreting cells (ASC). Cell suspensions containing putative ASC were first incubated with magnetic microbeads coated with antibodies specific for a given cell surface marker. After separation of bead-cell clusters and free cells, the resulting cell populations were examined for the presence of ASC by an ELISPOT assay. As a model system, the expression of selected cell differentiation markers by human circulating ASC has been evaluated after parenteral tetanus vaccination and during the course of a Leishmania infection. Prior treatment of blood MNC with beads coated with antibodies to CD38, HLA-DR or CD19 permitted the isolation of virtually all blood ASC. Further, prior immunomagnetic removal of T (CD2+) cells from blood MNC, followed by isolation of CD38+ cells facilitated the detection of Leishmania major-specific ASC in all six patients examined, whereas parasite-specific ASC among unfractionated blood mononuclear cells could only be detected in 3 out of these six patients. Simple and rapid, this approach provides not only accurate estimates of the frequency of ASC within a given B cell population or subpopulation, but can also efficiently enrich functional ASC from complex cell suspensions and thus should be particularly useful in situations where ASC are present at low frequencies.

Differential expression of tissue-specific adhesion molecules on human circulating antibody-forming cells after systemic, enteric, and nasal immunizations. A molecular basis for the compartmentalization of effector B cell responses.

Expression of the adhesion molecules CD44, L-selectin (CD62L), and integrin alpha 4 beta 7 by antibody-secreting cells (ASC) was examined in human volunteers after oral, rectal, intranasal, or systemic immunization with cholera toxin B subunit. Almost all blood ASC, irrespective of immunization route, isotype (IgG and IgA), and immunogen, expressed CD44. On the other hand, marked differences were observed between systemically and intestinally induced ASC with respect to expression of integrin alpha 4 beta 7 and L-selectin, adhesion molecules conferring tissue specificity for mucosal tissues and peripheral lymph nodes, respectively. Thus, most ASC induced at systemic sites expressed L-selectin, whereas only a smaller proportion of ASC expressed alpha 4 beta 7. In contrast, virtually all IgA- and even IgG-ASC detected after peroral and rectal immunizations expressed alpha 4 beta 7, with only a minor fraction of these cells expressing L-selectin. Circulating ASC induced by intranasal immunization displayed a more promiscuous pattern of adhesion molecules, with a large majority of ASC coexpressing L-selectin and alpha 4 beta 7. These results demonstrate that circulating ASC induced by mucosal and systemic immunization express different sets of adhesion molecules. Furthermore, these findings provide for the first time evidence for differential expression of adhesion molecules on circulating ASC originating from different mucosal sites. Collectively, these results may explain the anatomical division of mucosal and systemic immune responses in humans as well as the compartmentalization of mucosal immune responses initiated in the upper vs. the lower aerodigestive tract.

Multiple memory hearing aid. Consistency of program-usage in real-world listening situations.

The aim of this study was to assess the repeatability of the program-usage in everyday listening situations. A blind field test was used. The Widex Quattro (WQ) system served as a model for multiple memory hearing aids (linear amplification) Eleven experienced WQ wearers (41-73 years) with mild to moderate, recruiting, cochlear hearing losses participated. Eight of them regularly used all four available programs (all used at least three programs). The participants stated in duplicate the best hearing aid program in 15 real-world listening situations. The percentage of subjects who selected identical programs (repeatability) surpassed the level corresponding to pure guess under almost all listening conditions (14). Maximum repeatability (100%) was achieved by the five subjects who visited an industrial environment twice. Interestingly, the repeatability exceeded 70% in demanding listening situations such as: party, conversation in group, etc. Support for this high repeatability was given by a distinct improvement in the mean aided speech-to-noise threshold (3.7 dB, p < 0.005).

Polyamines inhibit myosin phosphatase and increase LC20 phosphorylation and force in smooth muscle.

The increase in Ca(2+)-activated force caused by polyamines in beta-escin-permeabilized guinda pig ileum is shown to be associated with increased myosin 20-kDa light chain (LC20) phosphorylation and shortening velocity. Myosin LC20 dephosphorylation with arrested kinase activity was slower in the presence of 1 mM spermine. Smooth muscle phosphatases (SMP-I, -II, -III, and -IV) isolated from turkey gizzard are all active against phosphorylated LC20, but only SMP-III and -IV dephosphorylate heavy meromyosin (HMM). Spermine inhibited SMP-III activity toward LC20 but stimulated HMM dephosphorylation, whereas SMP-IV was inhibited with both substrates. In contrast, SMP-I and -II were stimulated by spermine. The relative effects of different polyamines correlated with an increasing number of positive charges. Spermine did not affect binding of SMP-IV to myosin and did not dissociate any of the subunits of the enzyme. Incubation of permeabilized strips with SMP-IV resulted in attenuated responses to Ca2+, an effect that was opposed by spermine and abolished by microcystin-LR. We conclude that spermine selectively inhibits myosin phosphatase activity and suggest that polyamines function as endogenous myosin phosphatase inhibitors.