Plasmacytoid dendritic cells (pDCs) competent to make type I interferon were rigorously defined as a Ly-6C(+) and CD11c(Lo) subset of the B220(+)CD19(-) CD43(+)CD24(Lo) bone marrow (BM) Fraction A. Otherwise similar Ly6C(-) cells expressed the natural killer (NK) markers DX5 and NK1.1. pDCs represented a stable, discrete, and long-lived population. Stem cells and early lymphoid progenitors (ELPs), but not prolymphocytes, were effective precursors of pDCs, and their differentiation was blocked by ligation of Notch receptors. Furthermore, pDCs were present in the BM of RAG1(-/-), CD127/IL-7Ra(-/-), and Pax5(-/-) mice. pDCs in RAG1/GFP knock-in mice could be subdivided, and immunoglobulin D(H)-J(H) rearrangements, as well as transcripts for the B-lineage-related genes Pax5, mb1/CD79a, ebf, and Bcl11a, were identified only in the green fluorescent protein-positive (GFP(+)) pDC1 subset. All pDCs expressed terminal deoxynucleotidyl transferase (TdT), the ETS transcription factor Spi-B, the nuclear factor-kappaB transcription factor RelB, toll-like receptor 9 (TLR9), and interferon consensus sequence binding protein (ICSBP)/interferon regulatory factor 8 (IRF-8) transcripts; lacked CD16 and granulocyte colony-stimulating factor receptor (G-CSFR); and were uniformly interleukin-7 receptor alpha (IL-7Ralpha(-)) AA4.1(Lo), CD27(-), Flk-2(Lo), c-Kit(-), DX-5(-), and CD11b(-), while CD4 and CD8alpha were variable. GFP(+) pDC1 subset was less potent than GFP(-) pDC2s in T allostimulation and production of tumor necrosis factor alpha (TNFalpha), interferon alpha (IFNalpha), and interleukin-6 (IL-6), while only pDC2s made IFNgamma and IL-12 p70. Thus, 2 functionally specialized subsets of pDCs arise in bone marrow from progenitors that diverge from B, T, and NK lineages at an early stage.

We have used Southern's blotting technique to determine the extent to which the genes encoding the constant (C) regions of mu, alpha, gamma(1), and gamma(2b) immunoglobulin heavy (H) chains are altered in number and context from their germline (embryo) state in a series of 14 plasmacytomas expressing various H chain classes. In the three mu chain-producing plasmacytomas studied there was no evidence of rearrangement of C(H) genes other than C(mu). In contrast, rearrangement and deletion of nonexpressed C(H) genes was frequent in plasmacytomas that produce gamma or alpha chains. The observed pattern of deletions is consistent with the idea that the ontogenetic switch in H chain class requires C(H) gene deletion. Frequently, though not always, such deletions as well as other types of rearrangement occur in both allelic loci. Particularly noteworthy are three gamma(2a)-expressing tumors in which C(alpha) gene rearrangement is evident in both alleles. We incorporate these observations into a probabilistic model of B cell development: in the first phase, deletions may occur between the C(mu) gene and the variable (V(H)) gene array, which result in the formation of a productive fused V(H)-C(mu) gene. The cell may then enter a second phase, which allows deletions within the C(H) gene arrays of both homologous chromosomes. Some deletions juxtapose the expressed V(H) gene with a second C(H) gene and result in a H chain class switch; others delete or alter the context of C(H) genes without changing the phenotype of the cell. We predict that switching can be both a single-step and a multi-step process, and that in the latter case those rearrangements that do not result in a switch may be physiologically significant in that they may limit the options of further switching.

Early inflammatory events are initiated by phased production of C5a and interleukin-8 in tissue. Most serotypes of group A streptococci express a surface-bound peptidase (SCPA) which specifically cleaves mouse and human C5a chemotaxins. This study investigates the impact of SCPA on colonization of the nasopharyngeal mucosa of mice and evaluates its potential to induce protective immunity. Two strains, serotypes M6 and M49, which contain insertion and deletion mutations in the SCPA gene (scpA) and represent the two major subdivisions of group A streptococci, were characterized and compared in a mouse intranasal infection model. In this model, SCPA mutants were more rapidly cleared from the nasopharynges of inoculated mice compared with wild-type strains. A 2,908-bp fragment of scpAAAlthough the purified deltaSCPAAimmunoglobulin A (IgA) and serum IgG antibodies and reduced the potential of wild-type M1, M2, M6, M11, and M49 streptococci to colonize. These experiments suggest a new approach to vaccine development for prevention of streptococcal pharyngitis.

The neonatal Fc receptor, FcRn, transports immunoglobulin G (IgG) across cellular barriers between mother and offspring. FcRn also protects circulating IgG from catabolism, probably during transport across the capillary endothelium. Only one cell culture model of transcytosis has been used extensively, the transport of IgA from the basolateral to the apical surface of Madin-Darby canine kidney cells by the polymeric immunoglobulin receptor (pIgR). We report that rat inner medullary collecting duct (IMCD) cells transfected with DNA encoding the (alpha) subunit of rat FcRn specifically and saturably transport Fc when grown as polarized monolayers. Using this system, we have found that transcytosis by FcRn, like transcytosis by the pIgR, depends upon an intact microtubule system. FcRn differs most strikingly from the pIgR in its ability to transport its ligand in both the apical to basolateral and basolateral to apical directions. The phosphatidylinositol 3-kinase inhibitors wortmannin and LY294002 inhibited basolateral to apical transport by FcRn more than apical to basolateral transport, suggesting that there are differences in the mechanisms of transport in the two directions. Lastly, we found that transcytosis by FcRn depends upon vesicular acidification. We anticipate that the IMCD cell culture model will allow further elucidation of the mechanism of IgG transport by FcRn.

CC chemokine ligand 1 (CCL1; I-309) is a CC chemokine that interacts with CC chemokine receptor 8, which is preferentially expressed in polarized T helper cell type 2 and Tc2 cells, in eosinophils, and in T regulatory cells. The present study, prompted by transcriptional profiling of human monocytes undergoing different forms of activation, was designed to characterize the production of CCL1 in monocytes compared with the production of other chemokines (CCL2, CCL22, and CCL18) differentially regulated by distinct activation signals. Lipopolysaccharide (LPS), interferon-gamma (IFN-gamma), interleukin (IL)-1beta, tumor necrosis factor alpha, IL-4, IL-13, IL-10, IL-6, IL-18, and combinations thereof did not induce CCL1 production in monocytes, and some of these signals stimulated production of reference chemokines. Induction of CCL1 in monocytes required engagement of Fc receptor for immunoglobulin G (FcgammaR)II and exposure to IL-1beta or LPS. This combination of stimuli results in a form of M2 (M2b, Type 2) macrophage activation. FcgammaR engagement also induced CCL22 and amplified its stimulation by IL-4. In contrast, FcgammaR stimulation inhibited the IL-10- and LPS-mediated induction of CCL18. IL-10, IL-4, and IFN-gamma inhibited induction of CCL1 by FcgammaR ligation and IL-1beta. CCL1 was present in synovial fluids and macrophages in juvenile idiopathic arthritis. Thus, regulation of CCL1 in human monocytes is unique, with an obligate requirement of FcgammaR engagement and costimulation by signals (IL-1beta and LPS), which use the myeloid differentiation primary-response protein 88 adaptor protein. Thus, CCL1 is a CC chemokine with a unique pattern of regulation associated with a distinct form of M2 (Type 2, M2b) monocyte activation, which participates in macrophage-dependent regulatory circuits of innate and adaptive immunity.

Experimental evidence suggests a role for obesity in the formation and progression of some glomerular lesions, but data for human glomerulonephritis are lacking. In a cohort of 162 incident patients with biopsy-proven immunoglobulin A (IgA) nephropathy, we assessed whether the presence of an elevated body mass index (BMI>>/= 25 kg/m(2)) at the time of the first renal biopsy (RB1) correlated with clinical data at RB1 (24-hour proteinuria, arterial hypertension, and renal function), pathological data (global optical score [GOS] with detailed pathological indices), and clinical progression to both arterial hypertension and chronic renal failure (CRF). In both univariate and multivariate analyses, the presence of an elevated BMI at RB1 was significantly associated with the severity of pathological renal lesions (GOS and vascular, tubular, and interstitial indices). Hypertension-free survival was significantly less in overweight patients (P: < 0.0001) compared with those with normal weight. In a Cox regression analysis for hypertension-free survival including 24-hour proteinuria greater than 1 g, GOS, and metabolic parameters, only elevated BMI and GOS were independent factors for the development of arterial hypertension. CRF-free survival was also significantly less in patients with an excessive BMI. In a multivariate Cox regression analysis for CRF-free survival, hypertension, GOS, and BMI at RB1 were independent risk factors for CRF. In IgA nephropathy, excessive body weight and/or BMI are underestimated predictive factors for the development of arterial hypertension and, ultimately, CRF.

BACKGROUND

To date, drug response genes have not proved as useful in clinical practice as was anticipated at the start of the genomic era. An exception is in the treatment of chronic hepatitis C virus (HCV) genotype 1 infection with pegylated interferon-alpha and ribavirin (PegIFN/R). Viral clearance is achieved in 40%-50% of patients. Interleukin 28B (IL28B) genotype predicts treatment-induced and spontaneous clearance. To improve the predictive value of this genotype, we studied the combined effect of variants of IL28B with human leukocyte antigen C (HLA-C), and its ligands the killer immunoglobulin-like receptors (KIR), which have previously been implicated in HCV viral control.

RESULTS

We genotyped chronic hepatitis C (CHC) genotype 1 patients with PegIFN/R treatment-induced clearance (n = 417) and treatment failure (n = 493), and 234 individuals with spontaneous clearance, for HLA-C C1 versus C2, presence of inhibitory and activating KIR genes, and two IL28B SNPs, rs8099917 and rs12979860. All individuals were Europeans or of European descent. IL28B SNP rs8099917 "G" was associated with absence of treatment-induced clearance (odds ratio [OR] 2.19, p = 1.27×10(-8), 1.67-2.88) and absence of spontaneous clearance (OR 3.83, p = 1.71×10(-14), 2.67-5.48) of HCV, as was rs12979860, with slightly lower ORs. The HLA-C C2C2 genotype was also over-represented in patients who failed treatment (OR 1.52, p = 0.024, 1.05-2.20), but was not associated with spontaneous clearance. Prediction of treatment failure improved from 66% with IL28B to 80% using both genes in this cohort (OR 3.78, p = 8.83×10(-6), 2.03-7.04). There was evidence that KIR2DL3 and KIR2DS2 carriage also altered HCV treatment response in combination with HLA-C and IL28B.

CONCLUSIONS

Genotyping for IL28B, HLA-C, and KIR genes improves prediction of HCV treatment response. These findings support a role for natural killer (NK) cell activation in PegIFN/R treatment-induced clearance, partially mediated by IL28B.

Local synthesis seems to be decisive for the selective secretion of 19S immunoglobulin M into parotid secretions. The "transport piece" is apparently not involved in the secretion of immunoglobulin M, for there is no association between the two components. The possible significance of the normal association of transport piece with secreted immunoglobulin A remains to be clarified.

Macrophages from Tpl2 knockout (Tpl2(-/-)) mice exhibit a defect in ERK activation by lipopolysaccharide (LPS). This impairs the nucleocytoplasmic transport of the tumor necrosis factor alpha (TNF-alpha) mRNA and prevents the induction of TNF-alpha by LPS. As a result, Tpl2(-/-) mice are resistant to LPS/D-galactosamine-induced shock. We demonstrate that Tpl2 is essential for ERK signals transduced by members of the TNF receptor superfamily, such as CD40 and the TNF receptor 1. Thus, ERK activation was impaired in Tpl2(-/-) B cells and macrophages stimulated with agonistic CD40 antibody or TNF-alpha, whereas the induction of other mitogen-activated protein kinases, such as JNK and p38, and the activation of NF-kappaB were unaffected. Tpl2 was recruited to a CD40/TRAF6 complex in response to CD40 stimulation. Moreover, TRAF6, which when overexpressed activates ERK, failed to do so in Tpl2(-/-) cells. The selective signaling defect resulting from the inactivation of Tpl2 allowed us to demonstrate that CD40-mediated ERK activation contributes to immunoglobulin production but is not essential for B-cell proliferation.

Immunoglobulin A (IgA) induction primarily occurs in intestinal Peyer's patches (PPs). However, the cellular interactions necessary for IgA class switching are poorly defined. Here we show that in mice, activated B cells use the chemokine receptor CCR6 to access the subepithelial dome (SED) of PPs. There, B cells undergo prolonged interactions with SED dendritic cells (DCs). PP IgA class switching requires innate lymphoid cells, which promote lymphotoxin-β receptor (LTβR)-dependent maintenance of DCs. PP DCs augment IgA production by integrin αvβ8-mediated activation of transforming growth factor-β (TGFβ). In mice where B cells cannot access the SED, IgA responses against oral antigen and gut commensals are impaired. These studies establish the PP SED as a niche supporting DC-B cell interactions needed for TGFβ activation and induction of mucosal IgA responses.

Whey, a protein complex derived from milk, is being touted as a functional food with a number of health benefits. The biological components of whey, including lactoferrin, beta-lactoglobulin, alpha-lactalbumin, glycomacropeptide, and immunoglobulins, demonstrate a range of immune-enhancing properties. In addition, whey has the ability to act as an antioxidant, antihypertensive, antitumor, hypolipidemic, antiviral, antibacterial, and chelating agent. The primary mechanism by which whey is thought to exert its effects is by intracellular conversion of the amino acid cysteine to glutathione, a potent intracellular antioxidant. A number of clinical trials have successfully been performed using whey in the treatment of cancer, HIV, hepatitis B, cardiovascular disease, osteoporosis, and as an antimicrobial agent. Whey protein has also exhibited benefit in the arena of exercise performance and enhancement.

Malignant astrocytomas, which are highly invasive, vascular neoplasms, compose the majority of nervous system tumors in humans. Elevated expression of fibroblast growth factors (FGFs) in astrocytomas has implicated the FGF family of mitogens in the initiation and progression of astrocyte-derived tumors. In this study, we demonstrated that human astrocytomas undergo parallel changes in FGF-receptor (FGFR) expression during their progression from a benign to a malignant phenotype. FGFR type 2 (BEK) expression was abundant in normal white matter and in all low-grade astrocytomas but was not seen in malignant astrocytomas. Conversely, FGFR type 1 (FLG) expression was absent or barely detectable in normal white matter but was significantly elevated in malignant astrocytomas. Malignant astrocytomas also expressed an alternatively spliced form of FGFR-1 (FGFR-1 beta) containing two immunoglobulin-like disulfide loops, whereas normal human adult and fetal brains expressed a receptor form (FGFR-1 alpha) containing three immunoglobulin-like disulfide loops. Intermediate grades of astrocytic tumors exhibited a gradual loss of FGFR-2 and a shift in expression from FGFR-1 alpha to FGFR-1 beta as they progressed from benign to malignant phenotype. These results suggest that differential expression and alternative splicing of FGFRs may be critical in the malignant progression of astrocytic tumors.

Immunoglobulin A (IgA) provides protection against pathogens at mucosal surfaces. Chemotactic responses have been hypothesized to target IgA plasma cells involved in mucosal immune responses. We show here that thymus-expressed chemokine (TECK, CCL25) is a potent and selective chemoattractant for IgA antibody-secreting cells (ASC), efficiently recruiting IgA-producing cells from spleen, Peyer's patches, and mesenteric lymph node. Cells secreting IgA antibody in response to rotavirus, an intestinal pathogen, also respond well. In contrast, IgG- and IgM-ASC respond poorly. Epithelial cells in the small intestines, a principal site of IgA-ASC localization and IgA production in the body, highly and selectively express TECK. The migration of IgA-ASC to the intestinal epithelial cell chemokine TECK may help target IgA-producing cells to the gut wall, thus helping define and segregate the intestinal immune response.

The immune system of higher organisms is composed largely of two distinct cell types, B lymphocytes and T lymphocytes, each of which is independently capable of recognizing an enormous number of distinct entities through their antigen receptors; surface immunoglobulin in the case of the former, and the T-cell receptor (TCR) in the case of the latter. In both cell types, the genes encoding the antigen receptors consist of multiple gene segments which recombine during maturation to produce many possible peptides. One striking difference between B- and T-cell recognition that has not yet been resolved by the structural data is the fact that T cells generally require a major histocompatibility determinant together with an antigen whereas, in most cases, antibodies recognize antigen alone. Recently, we and others have found that a series of TCR V beta gene sequences show conservation of many of the same residues that are conserved between heavy- and light-chain immunoglobulin V regions, and these V beta sequences are predicted to have an immunoglobulin-like secondary structure. To extend these studies, we have isolated and sequenced eight additional alpha-chain complementary cDNA clones and compared them with published sequences. Analyses of these sequences, reported here, indicate that V alpha regions have many of the characteristics of V beta gene segments but differ in that they almost always occur as cross-hybridizing gene families. We conclude that there may be very different selective pressures operating on V alpha and V beta sequences and that the V alpha repertoire may be considerably larger than that of V beta.

In antibody responses, B cells switch from the expression of immunoglobulin (Ig) mu and delta heavy (H) chains to that of other Ig classes (alpha, gamma, or epsilon), each with a distinct effector function. Membrane-bound forms of alpha, gamma, and epsilon, but not mu and delta, have highly conserved cytoplasmic tails. Mutant mice unable to express membrane gamma1 H chains or producing tailless gamma1 H chains failed to generate efficient IgG1 responses and IgG1 memory. H chain membrane expression after class switching is thus required for these functions, and class switching equips the B cell antigen receptor with a regulatory cytoplasmic tail that naïve B cells lack.

Ly-1 B cells have the distinctive property of continuous self-replenishment and, as we have shown previously, can be further distinguished from conventional B cells on the basis of greatly elevated constitutive and inducible production of the recently described cytokine interleukin 10 (IL-10). To test the possibility that IL-10 acts as either an autocrine or paracrine growth factor for Ly-1 B cells, we treated mice continuously from birth to 8 wk of age with a monoclonal rat IgM antibody that specifically neutralizes mouse IL-10. Mice treated in this way lacked peritoneal-resident Ly-1 B cells, contained greatly reduced serum immunoglobulin M levels, and were unable to generate significant in vivo antibody responses to intraperitoneal injections of alpha 1,3-dextran or phosphorylcholine, antigens for which specific B cells reside in the Ly-1 B cell subset. In contrast, conventional splenic B cells of anti-IL-10-treated mice were normal with respect to total numbers, phenotype, and in vitro responsiveness to B cell mitogens and the thymus-dependent antigen trinitrophenyl-keyhole limpet hemocyanin (TNP-KLH). The mechanism of Ly-1 B cell depletion appeared to be related to elevation of endogenous interferon gamma (IFN-gamma) levels in anti-IL-10-treated mice, since coadministration of neutralizing anti-IFN-gamma antibodies substantially restored the number of peritoneal-resident Ly-1 B cells in these mice. These results implicate IL-10 as a regulator of Ly-1 B cell development, and identify a procedure to specifically deplete Ly-1 B cells, thereby allowing further evaluation of the role of these cells in the immune system.

OBJECTIVE

The specificity of colonization by Helicobacter pylori and complex host-bacterium interactions cannot be readily examined in humans. The aim of this study was to perform such analyses in rhesus monkeys.

METHODS

Four animals that had been cured of natural H. pylori colonization were challenged with a mixture of 7 strains of human origin, and bacteria recovered during periodic videogastroscopy were DNA fingerprinted.

RESULTS

Three animals carried mixtures of several strains for 4 months, after which strain J166 predominated. In the fourth animal, only strain J238 was isolated from the earliest phase of colonization through 7 months, but strain J166 again became predominant by 10 months after the challenge. Gastritis scores and plasma gastrin and anti-H. pylori immunoglobulin G titers reached levels observed in naturally colonized animals by 4 months after the challenge; however, no plasma immunoglobulin A response was observed up to 10 months.

CONCLUSIONS

These results show that (1) natural colonization does not elicit protective immunity against subsequent H. pylori challenge; (2) individuals differ in susceptibility to different H. pylori strains during initial stages of colonization; and (3) certain strains are better suited than others for long-term survival in different hosts. These observations show the complexity of H. pylori-host interactions.

Hepatitis C virus (HCV) is one of the leading causes of chronic liver diseases and B-lymphocyte proliferative disorders, including mixed cryoglobulinemia and B-cell lymphoma. It has been suggested that HCV infects human cells through the interaction of its envelope glycoprotein E2 with a tetraspanin molecule CD81, the putative viral receptor. Here, we show that the engagement of B cells by purified E2 induced double-strand DNA breaks specifically in the variable region of immunoglobulin (V(H)) gene locus, leading to hypermutation in the V(H) genes of B cells. Other gene loci were not affected. Preincubation with the anti-CD81 monoclonal antibody blocked this effect. E2-CD81 interaction on B cells triggered the enhanced expression of activation-induced cytidine deaminase (AID) and also stimulated the production of tumor necrosis factor alpha. Knockdown of AID by the specific small interfering RNA blocked the E2-induced double-strand DNA breaks and hypermutation of the V(H) gene. These findings suggest that HCV infection, through E2-CD81 interaction, may modulate host's innate or adaptive immune response by activation of AID and hypermutation of immunoglobulin gene in B cells, leading to HCV-associated B-cell lymphoproliferative diseases.

We investigated a patient (W.B.) with chronic symptomatic Giardiasis despite seven separate courses of either quinacrine or metronidazole who was cured by combined quinacrine and metronidazole. After isolating Giardia lamblia from W.B., we cultured the trophozoites to make the following observations. In vitro drug testing showed that (a) W.B.'s organisms were not more drug resistant than three other isolates and that (b) W.B.'s organisms were more sensitive to combined quinacrine and metronidazole than to either drug alone. Isolates of Giardia lamblia from W.B. and 3 other patients did not produce detectable enterotoxin in four different assays. W.B. had normal levels of circulating immunoglobulins, detectable intestinal immunoglobulin A, circulating immunoglobulin G anti-Giardia lamblia antibodies, and lymphocyte responsiveness to solubilized giardia lamblia. However, monocytes-macrophages from W.B. exhibited reduced killing for Giardia lamblia compared with normal subjects.

CONCLUSIONS

(a) The chronicity of our patient's infection was not due to the organism having unique properties of drug resistance. (b) Combined quinacrine and metronidazole, which cured our patient's chronic giardiasis, should be tried in patients in whom infection persists after single drug therapy. (c) The diarrhea in our patient was probably caused by a mechanism other than Giardia lamblia-induced secretion by currently recognized enterotoxins. (d) Reduced cellular cytotoxicity for Giardia lamblia may have contributed to the persistence of our patient's infection and should be suspected in other patients with chronic giardiasis.

The antigen receptor on human T lymphocytes consists of two variable immunoglobulin-like glycoproteins, alpha and beta, which occur in association with three invariable T3 membrane proteins. In humans two of these proteins, T3-gamma and T3-delta, are glycoproteins of relative molecular mass (Mr) 25,000 (25K) and 20,000 (20K), respectively, while the third, T3-epsilon, is a 20K non-glycosylated protein. On the surface of murine T cells, a non-glycosylated protein dimer composed of 17K subunits (T3-zeta) is found associated with the T-cell receptor alpha and beta chains and the three T3-like polypeptide chains. It is generally accepted that major histocompatibility complex-restricted antigen recognition is a function of the alpha-beta heterodimer. This has led to the postulation that the proteins of the T3 complex are involved in the signal transduction that immediately follows antigen recognition via the antigen receptor. Events believed to be involved in early T-cell activation, such as rapid increases in phosphatidylinositol turnover and free intracellular calcium, can be triggered by antibodies directed against either the T3 complex or the clonotypic receptor. We have previously reported our findings on the cloning of the complementary DNA and genomic structure encoding both the human and murine 20K glycoprotein, T3-delta (refs 11-13). We now present our results on the cloning of the cDNA encoding the human 20K non-glycosylated chain, T3-epsilon.

Three restriction points control the cell cycle of activated B lymphocytes. The first occurs directly after mitosis and is controlled by the occupancy of surface-bound immunoglobulin. The second is observed approximately 4 h after mitosis in the G1 phase of the cycle, that is, before DNA replication, and is controlled by growth factors that are produced by macrophages which we have previously classified as alpha-type factors. The third restriction point occurs in the G2 phase, 2-4 h before mitosis, and is controlled by beta-type growth factors probably produced by helper T lymphocytes. The third component of complement, C3, has long been implicated in the control of B-cell responses. C3 is secreted by monocytes and macrophages. We have found recently that crosslinked, but not soluble, human C3 stimulates activated, but not resting, murine B cells to thymidine uptake. Here we investigate the role of C3b and C3d in the progression of the cell cycle of activated, synchronized murine B cells. We find that crosslinked C3d replaces the action of alpha-factors within the cell cycle of these cells and allows entry into S phase. In contrast, soluble C3d inhibits the action of alpha-factors. This implies that a C3d-specific receptor, probably the murine analogue to the human complement receptor CR2, is a growth factor receptor on activated B cells that will give the cell a growth-positive signal when it is crosslinked, while occupancy by the soluble form of C3d will result in inhibition of the action of alpha-factors or of crosslinked C3b or C3d. A stretch of weak homology between the cDNA sequence of murine C3d and those of murine growth factors indicates that an insulin-like growth factor could be the active principle of C3d that controls the cell cycle of activated B cells.

Of 20 fibroblast cell strains from patients with osteogenesis imperfecta (OI), a disease caused by mutations in the genes encoding type I procollagen, three had increased synthesis of BiP (GRP78), an hsp70-related, endoplasmic reticulum-resident protein. All three strains carry unique mutations in pro alpha 1(I) chains which impair type I procollagen chain association. Immunoprecipitation and pulse-chase experiments show that BiP (immunoglobulin heavy chain-binding protein) stably binds pro alpha 1(I) chains in these three cell strains after a brief lag. Ascorbate, which increases procollagen synthesis, increases BiP synthesis and content in these three strains and not in the others. In one of these three strains, BiP content is constitutively elevated prior to ascorbate treatment, and BiP is less inducible. This strain also has relatively high levels of synthesis and content of GRP94, another endoplasmic reticulum-resident stress protein. Pretreating each of the three cell strains to increase their BiP content reduces subsequent ascorbate-mediated BiP induction. BiP synthesis in the 17 other OI strains examined, which had a variety of type I procollagen mutations, was normal. These results suggest that BiP is induced by and binds procollagen with specific types of mutations: ones in the carboxyl-terminal propeptide that interfere with chain association. The recognition by BiP of such procollagen in OI cell strains shows that BiP plays a role in the physiological response to the production of some disease-producing abnormal proteins.

The chemokine receptor, CXCR4, is expressed by human melanomas, and its ligand, CXCL12, is frequently produced at sites of melanoma metastasis. Herein, we examine CXCR4-enhanced binding of B16 murine melanoma cells to endothelial cells (ECs) and recombinant adhesion molecules in vitro to determine the role of tumor- and EC-derived adhesion molecules in tumor metastasis. By flow cytometry, unstimulated primary lung ECs showed constitutive expression of vascular cellular adhesion molecule 1 (VCAM-1), whereas skin-derived ECs did not. All B16 cell lines tested showed constitutive expression of alpha(4) and beta(1) integrin chains but showed no expression of beta(2) integrins. CXCR4-B16 arrest on VCAM-1/immunoglobulin-coated plates and tumor necrosis factor alpha-stimulated ECs under physiological shear stress conditions (1.5 dynes/cm(2)) was rapid, resistant to shear stress of 10 dynes/cm(2), and showed no evidence of rolling before arrest. In vitro, CXCR4-B16 cell binding to ECs was blocked by anti-beta(1) and anti-CXCL12 monoclonal antibodies. In vivo, metastasis of CXCR4-B16 cells to murine lungs was strongly inhibited by anti-CXCL12 and two different anti-beta(1) monoclonal antibodies. Finally, CXCR4-B16 exposed to CXCL12 rapidly increased binding affinity for soluble VCAM-1/immunoglobulin as detected by a flow cytometric assay. Thus, beta(1) integrins play a critical role in CXCR4-mediated B16 tumor cell metastasis in vivo and may be a potential target for inhibition of tumor metastasis, particularly to the lung.

Milk is the single source of nutrients for the newborn mammal. The composition of milk of different mammals has been adapted during evolution of the species to fulfill the needs of the offspring. Milk not only provides nutrients, but it also serves as a medium for transfer of host defense components to the offspring. The host defense proteins in the milk of different mammalian species are expected to reveal signatures of evolution. The aim of this study is therefore to study the difference in the host defense proteome of human and bovine milk. We analyzed human and bovine milk using a shot-gun proteomics approach focusing on host defense-related proteins. In total, 268 proteins in human milk and 269 proteins in bovine milk were identified. Of these, 44 from human milk and 51 from bovine milk are related to the host defense system. Of these proteins, 33 were found in both species but with significantly different quantities. High concentrations of proteins involved in the mucosal immune system, immunoglobulin A, CD14, lactoferrin, and lysozyme, were present in human milk. The human newborn is known to be deficient for at least two of these proteins (immunoglobulin A and CD14). On the other hand, antimicrobial proteins (5 cathelicidins and lactoperoxidase) were abundant in bovine milk. The high concentration of lactoperoxidase is probably linked to the high amount of thiocyanate in the plant-based diet of cows. This first detailed analysis of host defense proteins in human and bovine milk is an important step in understanding the function of milk in the development of the immune system of these two mammals.