Protective effect of rSm28GST-specific T cells in schistosomiasis: role of gamma interferon.

Immunization with a single dose of 50 micrograms of recombinant Schistosoma mansoni 28-kDa glutathione-S-transferase (rSm28GST) was able to induce a reduction in the worm burden, the number of eggs, and the degree of hepatic fibrosis as quantified by the measurement of collagen content in the liver of S. mansoni-infected mice. No relationship was found between anti-Sm28GST immunoglobulin G and immunoglobulin A titers and the levels of protection obtained. Adoptive transfers of Sm28GST-specific total, CD4+, or CD8+ T cells reproduced the protective effect obtained with the recombinant molecule. Moreover, experiments studying in vivo T-cell depletion demonstrated that anti-CD4- or anti-CD8-treated mice showed a significant decrease in the protective effect conferred, suggesting a role of the two T-cell subpopulations in the expression of Sm28GST-mediated protection against hepatic damage. Sm28GST-specific cells produced little interleukin-4 and high levels of gamma interferon. Treatment of immunized mice with anti-gamma interferon antibody totally suppressed the Sm28GST-induced protective effect and led to the rapid death of infected animals, suggesting a role for this cytokine in the expression of the protective immunity obtained after immunization with rSm28GST.     

GM-CSF and TNF-alpha synergize to increase in vitro granuloma size of PBMC from humans induced by Schistosoma mansoni recombinant 28-kDa GST

The 28-kDa Glutathione S-transferase of Schistosoma mansoni (Sm28 GST) was described as a protective antigen capable of reducing female fecundity and the number of eggs in mice hepatic tissues. The role of GM-CSF and TNF-alpha in the in vitro granuloma reaction of peripheral blood mononuclear cells (PBMC) from chronic intestinal schistosomiasis patients before and after chemotherapy treatment to S. mansoni recombinant Sm28 GST was evaluated. Treatment of PBMC with recombinant Sm28 GST caused a significant increase in granuloma formation when compared to SEA or SWAP. Contrary to SEA or SWAP, Sm28 GST was not capable of inducing significant cellular proliferation. Moreover, recombinant Sm28 GST promoted a significant elevation in GM-CSF and TNF-alpha levels. However, we did not detect any significant IL-10 production. When Sm28 GST was applied in the presence of anti-GM-CSF or anti-TNF-alpha antibodies in cultures, we observed a significant decrease in granuloma size. Indeed, our results demonstrated that Sm28 GST was capable of promoting high in vitro granuloma index, and this event was associated with the balance of GM-CSF and TNF-alpha. These evidences suggest a role for GM-CSF as a major mediator in increasing granuloma reaction in human schistosomiasis. This event may contribute to exacerbate the pathology resulting from egg deposition in host tissues.     

H-2 linked immune response to murine experimental Schistosoma mansoni infections.

Mice of two congenic inbred strains C3H/Sn (H-2k) and C3H.B10 (H-2b) were infected with 100 Schistosoma mansoni cercariae. After the infection, the following parameters for the immunological response were studied: worm burden, mortality, antibody titre, spleen index, eosinophilia, delayed type of hypersensitivity and in vitro response to three S. mansoni antigen preparations. No difference in the worm burden and in the in vitro response to the antigen preparations of adult worm antigen, soluble egg antigens and the egg antigen MSA1, was found. The C3H.B10 mice showed a significantly higher mortality, antibody titre and delayed type of hypersensitivity while the C3H/Sn mice showed asignificantly higher spleen index and eosinophilia. This indicates that the H-2 region influences the course of an acute S. mansoni infection, whereas the susceptibility to the infection seems not to be influenced, as is shown by the worm burden.     

青蒿琥酯对曼氏血吸虫雌虫生殖作用的影响

目的 :通过观察青蒿琥酯对曼氏血吸虫雌虫产卵的影响 ,分析药物的抗生殖作用。方法 :小鼠尾部接触感染曼氏血吸虫尾蚴后口服不同剂量青蒿琥酯 ,灌流后收集虫体 ,记数。解剖小鼠取出肝、肠 ,组织溶解后计数虫卵 ,分析药物作用后雌虫平均产卵量的变化 ;收集无损虫体进行体外培养 ,计数雌虫体外产卵并观察虫卵形态。结果 :在 30 0mg kg和 5 0 0mg kg给药组小鼠的肝、肠中未查到虫卵 ,10 0mg kg给药组小鼠的雌虫平均产卵量与对照组存在极显著性差异 ;体外培养结果中 ,30 0mg kg和 5 0 0mg kg给药组的虫体未见产卵 ,10 0mg kg给药组雌虫平均产卵量与对照组存在显著差异。显微观察发现 10 0mg kg给药组雌虫体外培养所产的虫卵多外附泡状物 ,侧棘受损 ,虫卵结构异常。结论 :青蒿琥酯能降低或完全抑制存活雌虫的平均产卵量、导致雌虫异常卵的产生 ,具有抗曼氏血吸虫雌虫生殖的作用 ,能有效预防曼氏血吸虫病的发生和传播     

Role of IgE in primary murine Schistosomiasis mansoni

Schistosoma mansoni infection proceeds in normal mice in the absence of detectable levels of polyclonal or specific immunoglobulin (Ig)E until worms mature and deposit eggs. Hence, the course of a primary S. mansoni infection is not expected to vary appreciably in mice with defects in the IgE production. Experimental increase of IgE production early after infection may, however, influence worm development. In the first approach towards this goal, BALB/c mice were injected with interleukin(IL)4 to raise the level of endogenously synthesized IgE. A significant increase in serum polyclonal IgE and antischistosome IgG1 during the prepatent period was not associated with significant changes in worm and egg burden or liver pathology. During the second approach, mice were injected with IgE which was affinity purified from serum of BALB/c mice infected for 16 weeks with S. mansoni. The purified IgE bound to carbohydrate-independent epitopes of soluble antigens from 3 h larvae, adult worms and eggs and recognized the schistosomular surface membrane. No differences in worm and egg load or granuloma number and size were noted between untreated and exogenous IgE-injected mice. Together, the data demonstrate that by itself IgE does not influence the outcome of infection in primary murine S. mansoni.     

Expression of a Schistosoma mansoni 28-kilodalton glutathione S-transferase in the livers of transgenic mice and its effect on parasite infection.

Schistosomiasis is a debilitating tropical disease for which an effective vaccine is needed. A 28-kDa glutathione S-transferase from Schistosoma mansoni (Sm28GST) has been shown to induce protective immunity. Sm28GST possesses significant sequence identity to mammalian GST isoforms. In order to study self-reactivity in mice immunized with Sm28GST and the concomitant phenomena of immune tolerance and epitope suppression, as well as their consequences for the protective immunity induced by this vaccination, we developed transgenic (Tg) mice that express Sm28GST under the control of a part of the mouse transferrin gene promoter. A study of (P28)Tg mice showed that the expression of Sm28GST was strictly localized in pericentrolobular hepatocytes. No histological change, inflammatory infiltrates, or modification of seric L-aspartate: 2-oxoglutarate aminotransferase concentration was observed over an 18-month period, despite a cross-reactivity between Sm28GST and a mouse molecule of 30 kDa. The immunoglobulin G anti-Sm28GST response of (P28)Tg mice immunized with recombinant Sm28GST was lower (P < 0.001) than that observed in non-(P28)Tg littermates and inversely proportional of Sm28GST liver expression. The response of non-(P28)Tg mouse spleen cells to Sm28GST stimulation was greater (P < 0.01) than that observed with (P28)Tg mouse spleen cells. (P28)Tg mice infected with 40 S. mansoni furcocercariae harbored more worms (P < 0.05) than did non-(P28)Tg control mice. The increase in the level of infection in (P28)Tg mice was reflected in concomitant increases in the numbers of adult worms and schistosome eggs found in livers and intestines after whole-body perfusion at 56 days postinfection, but no relative increase in the fertility of individual female worms was observed. The results obtained argue for the involvement of Sm28GST in reducing levels of infection and support the view that this enzyme has a central role in the maintenance of parasite viability, at least during its migration through host tissues.     

Schistosoma mansoni antigens modulate the activity of the innate immune response and prevent onset of type 1 diabetes

Infection with Schistosoma mansoni (S. mansoni) or exposure to eggs from this helminth inhibits the development of type 1 diabetes in NOD mice. In this study we show that soluble extracts of S. mansoni worm or egg completely prevent onset of type 1 diabetes in these mice but only if injection is started at 4 weeks of age. T cells from diabetes-protected mice make IL-10 in recall responses to parasite antigens. These cells are furthermore impaired in their ability to transfer diabetes to NOD-SCID recipients. Bone marrow dendritic cells derived from NOD mice are found to make more IL-10 and less IL-12 following culture with S. mansoni soluble egg antigens in conjunction with lipopolysaccharides. NOD mice are deficient in NKT cells. Soluble worm and egg antigens increase the numbers of V alpha 14i NKT cells in NOD mice. These effects of schistosome antigens on the innate immune system provide a mechanism for their ability to prevent type 1 diabetes in NOD mice.     

Ellagic acid reduces murine schistosomiasis mansoni immunopathology via up-regulation of IL-10 and down-modulation of pro-inflammatory cytokines production

Context: The main immunopathology in schistosomiasis mansoni consists of a granulomatous inflammatory and fibrosing reaction in the liver and intestine against tissue trapped parasite eggs, which is mediated by CD4^+?T cells. Ellagic acid (EA), a natural phenolic compound found in fruits and nuts, has potent anti-oxidant and anti-inflammatory properties.

Objective: The aim of the present study was to evaluate the potential effect of EA in the treatment of murine schistosomiasis mansoni and its induced immunopathology.

Materials and methods: Mice were infected, each with 40 Schistosoma mansoni (S. mansoni) cercariae and treated with EA at a total dose of 600?mg/kg body weight. At week eight of infection, mice were sacrificed; worm and egg burden were estimated; hepatic granuloma volume and collagen fibers deposition were evaluated; splenocytes were prepared and cultured in the presence of S. mansoni antigens.

Results: EA treatment did not show any significant effect on worm or egg burden. However, hepatic granuloma volume and collagen fibers deposition were largely reduced with EA treatment. EA treatment augmented specific IL-10 production in response to S. mansoni antigenic stimulation. However, specific IL-1β, IL-4, IL-12, IL-13, IL-17A, TNF-α and IFN-γ production were significantly reduced with ex vivo and in vivo EA treatment. Serum IgM and IgG levels significantly increased, whereas specific IgA and IgE levels did not significantly change with EA treatment.

Conclusion: EA treatment modulates cellular and humoral immune responses of infected mice and leads to a significant reduction of liver pathology in acute murine schistosomiasis mansoni.     

Induction of Cytotoxic T-Cell Activity by the Protective Antigen of Schistosoma mansoni Sm28GST or its Derived C-Terminal Lipopeptide

In a previous work the authors demonstrated that immunization with Schistosoma mansoni 28-kDa glutathion-S-transferase (Sm28GST) was able to reduce hepatic damage in infected mice and that the adoptive transfer of Sm28GST-specific T cells reproduced the protective effect obtained with the recombinant molecule. In the present paper, the authors show that Sm28GST is also able to stimulate an antigen-specific, cytotoxic T-cell response against Sm28GST-pulsed P815 target cells in normal mice and that effector cells induced in vivo were classical Class I MHC-restricted CD8⁺ lymphocytes. The authors found no spontaneous CTL activity against Sm28GST-pulsed target cells during the course of the infection by S. mansoni although Sm28GST is expressed at different developmental stages of the parasite. It was observed, however, that immunization with Sm28GST is sufficient to elicit a significant level of CTL response for 6 weeks in infected mice. The role of these Class I MHC-restricted CD8⁺ lymphocytes in the protection observed precisely at the same period in immunized mice remains to be elucidated. The authors also observe that immunization with the lipopeptidic form of the C-terminal peptide of the molecule (190–211 peptide) led to a CTL activation comparable to that observed after immunization with the whole molecule demonstrating the feasibility of using a synthetic lipopeptide as immunogen for a CTL response against Sm28GST epitopes. Moreover, like Sm28GST-specific CTLs, 190–211 lipopeptide-specific cells were also Class I MHC-restricted lymphocytes.     

Effects of sex and age on the susceptibility of C57BL/6J mice to infection with <Emphasis Type="Italic">Brachylaima cribbi</Emphasis> and the course of infection in NOD SCID mice

The C57BL/6J strain of Mus musculus is susceptible to the terrestrial trematode Brachylaima cribbi. The duration of infection in these mice is generally 9-12 weeks with a peak excretion of eggs at 4 weeks post-infection (wpi). The effects of age and sex on the course of infection were investigated by comparing infections in male and female mice aged 8 or 28 weeks at the time of infection. There were no significant differences in the susceptibility of the adolescent mice of either sex or older male mice. However, older, mature female mice were significantly more resistant to B. cribbi infection than older mature males and adolescent females with reduced worm burden, fecundity and egg fertility. In comparison with young males, all three parameters were again reduced but this was only significant statistically for reduced egg fertility. It is likely that mature female sex hormones influence resistance to B. cribbi infection. The susceptibility of immunodeficient NOD SCID mice was evaluated and compared with C57BL/6J mice. NOD SCID mice were susceptible to B. cribbi infection with the infection persisting with a relatively unchanged worm burden for the life of the mouse with the longest surviving mice being 31 wpi. The life-span of B. cribbi is therefore at least 31 weeks. There were no significant differences in egg excretion, worm burden or fecundity among NOD SCID mice at 4, 8 or 18 wpi. As the infection progressed in NOD SCID mice, the location of worms in the small intestine moved from the anterior third in the early stages of the infection to the mid- to posterior intestine in the later stages. Comparison of the infection in NOD SCID mice with C57BL/6J mice indicates that the expulsion of worms in the latter is mediated by an immune response.     

The effects of interleukin (IL)-12 and IL-4 deficiency on worm development and granuloma formation in Schistosoma japonicum-infected mice

CD4⁺ T-helper (Th) cell is widely recognized to be capable of influencing worm development and egg granuloma formation after schistosome infection. Interleukin (IL)-12 and IL-4 play key roles in regulation of Th cell differentiation. In the present study, we subcutaneously inoculated mice with hybridoma cells secreting monoclonal antibodies to neutralize IL-12 and IL-4 and explored the effects of IL-12 and IL-4 deficiency on the worm development and granuloma formation in mice infected with cercariae of Schistosoma japonicum. It was found that deficiency of host IL-12 and IL-4 supported normal parasite survival and fecundity. However, worm development (length and female fecundity) was significantly enhanced in anti-IL-12-treated mice. Mean length of worms in anti-IL-12-treated group was significantly greater than that of intact controls on day 28 after infection (females, 11.84 ± 1.20 mm vs. 9.45 ± 1.34; males, 9.35 ± 1.21 mm vs. 8.10 ± 0.85 mm, p < 0.05). Liver egg load per pair of worms (1,770.12 ± 470.67 vs. 806.08 ± 232.37, p < 0.05) and uterine egg load of ovigerous females (93.08 ± 27.85 vs. 46.05 ± 34.24, p < 0.05) in anti-IL-12-treated mice were significantly higher than those in intact control 28 days postinfection. But these effects diminished 42 days postinfection (p > 0.05). Granuloma size in anti-IL-12-treated mice was significantly larger than that in intact mice 42 days postinfection (398.3 ± 80.7 μm vs. 294.4 ± 72.2 μm, p < 0.05). Granuloma fibrosis dramatically intensified in anti-IL-12-treated mice but diminished in anti-IL-4-treated mice. The results suggest that IL-12 may play an impeditive role in the development of S. japonicum and in granuloma formation as well as fibrosis. IL-4 may promote granuloma formation but have no effect on worm development.     

Expression of a 28-kilodalton glutathione S-transferase antigen of Schistosoma mansoni on the surface of filamentous phages and evaluation of its vaccine potential

A cloning and expression system that allows display of proteins on the surface of filamentous phages was exploited to display a 28-kDa glutathione S-transferase (Sm28GST) antigen of the human parasite Schistosoma mansoni. The phage-displayed Sm28GST (pdGST) was immunoreactive and was recognized by immune sera, suggesting that the Sm28GST protein displayed on the surface of phages potentially maintains native conformation. Subsequent immunization studies showed that mice can develop high titers of antibodies against pdGST and do not require any additional adjuvant for immunization. Isotype analysis suggested that the pdGST immunization predominantly induced immunoglobulin G2b (IgG2b), IgG3, and IgM anti-GST antibodies in mice. Furthermore, the pdGST immunization was found to confer about 30% protection after a challenge infection with 100 cercariae of S. mansoni in BALB/c mice. These findings suggest that phage display is a simple, efficient, and promising tool to express candidate vaccine antigens for immunization against infectious agents.     

Molecular cloning and tissue distribution of a 26-kilodalton Schistosoma mansoni glutathione S-transferase

A Schistosoma mansoni cDNA library was constructed from the mRNA of adult worms in the expression vector lambda gt11 and screened with a rabbit antiserum raised against the 26-kDa S. mansoni glutathione S-transferase isoforms (Sm GST 26). Two clones were selected and the nucleotide sequences deduced. The predicted amino acid sequence, specified by these cDNAs, shows strong homology with a Schistosoma japonicum 26 kDa glutathione S-transferase and a lower level of homology with mammalian glutathione S-transferase class mu isoenzymes (EC 2.5.1.18). No significant homology score was found with a 28-kDa S. mansoni glutathione S-transferase (Sm GST 28). A study of the tissue distribution of the cloned Sm GST 26 by immunoelectron microscopy shows similarities to Sm GST 28 in that they are present in the tegument and in subtegumentary parenchymal cells. However, a major difference exists in the protonephridial region in which Sm GST 26 is present in the cytoplasmic digitations localized in the apical chamber delineated by the flame cell body, suggesting that Sm GST 26 may be actively excreted by adult worms.     

Comparison of the cloned genes of the 26- and 28-kilodalton glutathione S-transferases of Schistosoma japonicum and Schistosoma mansoni.

Both Schistosoma japonicum and S. mansoni contain 28- and 26-kDa glutathione S-transferases (GSTs). Despite their immunological cross-reactivity using rabbit antisera, the S. japonicum 28-kDa GST (Sj28) is weakly immunogenic relative to the S. mansoni protein (Sm28) in mouse immunization experiments using GSTs purified from adult worms. The difference in immunogenicity is also observed during schistosome infection in mice. Using surface-labeled living S. japonicum worms, evidence was obtained for a surface location of Sj28 comparable to that reported for the S. mansoni molecule. The nucleotide and deduced amino acid sequences of cDNA clones corresponding to Sj28 and Sm28 were compared. Despite obvious homology (77% identity), differences were found in regions known to contain T epitopes in the S. mansoni protein which may be an explanation for the striking differences in immunogenicity in regard to antibody production in mice. The 26-kDa GSTs of these two parasites (Sj26 and Sm26) are also closely related on the basis of nucleotide and deduced amino acid sequences, there being 82% identity in the putative coding regions. When the amino acid sequences of Sj28 and Sm28 were compared with those of Sj26 and Sm26, the overall sequence identity was approximately 20%. However, a relatively conserved region was identified in otherwise structurally different molecules which may participate in common properties of these enzymes.     

Schistosoma mansoni: unisexual infections sensitize mice for granuloma formation around intravenously injected eggs

 Mice carrying unisexual infection with male or female Schistosoma mansoni for 9 weeks developed accelerated and augmented reactions to S. mansoni eggs injected intravenously. The size of circumoval granulomas observed in the lungs of unisexually infected mice did not differ significantly from the reactions seen in bisexually infected mice. Tissue eosinophilia in the granulomas was also augmented similarly over that in naive mice by unisexual or bisexual infection. The cross-reactivity between worm and egg antigens is relevant to the development of acute toxemic schistosomiasis mansoni and, perhaps, to the consideration of antigens to be used for vaccination against S. mansoni infection. Received: 20 February 1996 / Accepted: 2 August 1996     

Relationship of Impairment of Schistosome 28-Kilodalton Glutathione S-Transferase (GST) Activity to Expression of Immunity to Schistosoma mattheei in Calves Vaccinated with Recombinant Schistosoma bovis 28-Kilodalton GST

Sera from calves vaccinated with the recombinant Schistosoma bovis-derived 28-kDa glutathione S-transferase (28GST) and subsequently naturally or experimentally exposed to Schistosoma mattheei were studied for their content of specific immunoglobulin G (IgG) and IgA antibodies to recombinant S. bovis 28GST as well as for their capacity to inhibit the enzymatic activity of the antigen. The results were analyzed in regard to the presence (natural infection) or absence (experimental infection) of a protective effect(s) (reductions in worm burden, egg load, fecal egg counts, and excretion of viable eggs) toward S. mattheei challenge. Under such conditions, no differences in the IgG- and IgA-specific antibodies to recombinant S. bovis 28GST or in the ability to block the catalytic function of the antigen between the two groups were recorded. Nevertheless, correlation analysis between the specific antibody responses to recombinant S. bovis 28GST and the inhibition of GST activity suggested an association with IgG in experimentally infected vaccinated animals, while in naturally infected vaccinated calves, the inhibitory activity appeared to be linked to a greater degree with IgA. These results suggest that in contrast to schistosomiasis in humans, IgG antibodies in calves with schistosomiasis may exhibit inhibitory functions toward GST enzymatic activity or have a modulatory effect on IgA antibody properties. Furthermore, sera from animals immunized with recombinant S. bovis 28GST recognized the native S. mattheei 28GST and achieved comparable levels of inhibition of activity of recombinant S. bovis 28GST and S. matthei 28GST, indicating the presence of cross-reactive epitopes on these two molecules.     

Obtention of a human primary humoral response against schistosome protective antigens in severe combined immunodeficiency mice after the transfer of human peripheral blood mononuclear cells

Peripheral blood mononuclear cells (PBMC) from healthy donors were injected into C.B.-17 severe combined immunodeficiency (scid) mice which were subsequently immunized with crude Schistosoma mansoni adult worm antigenic preparation (SWAP) or with recombinant S. mansoni 28-kDa glutathione transferase (r-Sm-28-GST) antigen. PBMC from a S. mansoni-infected patient were also transferred. The specific human anti-SWAP and anti-Sm-28-GST antibody responses were monitored. The presence in both cases of human specific antibodies in scid mouse sera was determined by enzyme-linked immunosorbent assay and Western blotting techniques using anti-human immunoglobulin reagents. No antibodies were detected in these sera using anti-mouse immunoglobulin antisera. These antibodies were functional since a cytotoxic activity against schistosomula was observed when monocytes were incubated with scid mouse sera positive for anti-Sm-28-GST antibodies.     

The C-type lectin SIGNR1 binds Schistosoma mansoni antigens in vitro, but SIGNR1-deficient mice have normal responses during schistosome infection

The de novo immune response to infectious organisms arises from the innate recognition of pathogen-associated molecular patterns (PAMPs) by the host's pattern recognition receptors (PRRs). As the generation of type 2 cytokine responses by the human trematode parasite Schistosoma mansoni is glycan mediated, there is a particular potential role for a C-type lectin receptor (CLR) to mediate the innate recognition of schistosome PAMPs. One such CLR, dendritic cell-specific intracellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN; CD209), has been shown to recognize glycans expressed by S. mansoni eggs. We show that SIGNR1 (SIGN-related 1; CD209b), a murine homologue of DC-SIGN that is expressed on macrophages, also binds both schistosome-soluble egg antigens and worm antigens in vitro. The generation of schistosome egg-induced pulmonary egg granulomas was not altered in SIGNR1-deficient mice. Following S. mansoni infection, the SIGNR1-deficient mice had an unaltered phenotype with an intact immunological response and no difference in pathology. In this study we demonstrate that although SIGNR1 recognizes S. mansoni antigens in vitro, this CLR is redundant during infection. This study highlights the finding that although there was binding of SIGNR1 to immunogenic factors produced in the S. mansoni life cycle, this recognition does not translate to a functional in vivo role for the PRR during infection.     

Interleukin-13 fusion cytotoxin arrests Schistosoma mansoni egg-induced pulmonary granuloma formation in mice

Schistosoma mansoni egg-induced lung pathology requires the actions of interleukin (IL)-4 and IL-13. Because receptors for IL-4 and IL-13 share chains, we examined the effect of a fusion protein comprised of IL-13 and Pseudomonas exotoxin (IL13-PE) on the development of pulmonary granulomas in mice. At day 8 after an intravenous injection of live S. mansoni eggs, whole lung samples from IL13-PE-treated mice exhibited significantly lower IL-4 and IL-13 gene expression, smaller granulomas, decreased collagen levels, and increased IL-13 receptor alpha2 gene expression compared to controls. The therapeutic effects of IL13-PE were also observed at day 16 despite the termination of IL13-PE treatment at day 8. These studies demonstrate that targeting IL-4- and IL-13- responsive cells with IL13-PE effectively arrests S. mansoni egg granuloma formation.