Management of Hepatic Angiomyolipoma

Preoperative diagnosis of hepatic angiomyolipoma is difficult, and the treatment for it remains controversial. The aim of this study is to review our experience in the treatment of hepatic angiomyolipoma and to propose a treatment strategy for this disease. We retrospectively collected the clinical, imaging, and pathological features of patients with hepatic angiomyolipoma. Immunohistochemical studies with antibodies for HMB-45, actin, S-100, cytokeratin, vimentin, and c-kit were performed. Treatment experience and long-term follow-up results are summarized. During a period of 9 years, 10 patients with hepatic angiomyolipoma were treated at our hospital. There was marked female predominance (nine patients). Nine patients received surgical resection without complications. One patient received nonoperative management with biopsy and follow-up. One patient died 11 months after surgery because of recurrent disease. We propose all symptomatic patients should receive surgical resection for hepatic angiomyolipoma. Conservative management with close follow-up is suggested in patients with asymptomatic tumors and meet the following criteria: (1) tumor size smaller than 5 cm, (2) angiomyolipoma proved through fine needle aspiration biopsy, (3) patients with good compliance, and (4) not a hepatitis virus carrier.
Introduction
Hepatic angiomyolipoma (AML) is a rare mesenchymal tumor of the liver composed of smooth muscle cells, adipose tissue, and proliferating blood vessels. Since its first description by Ishak in 1976, approximately 200 cases have been reported in the English literature.1 This type of tumor is usually seen in kidneys associated with tuberous sclerosis.2 Definite pathologic diagnosis is made by identification of the three different components and HMB-45 positive staining.3
In the past, this tumor has been considered an entirely benign and slow-growing lesion without the possibility of malignant transformation. Therefore, several authors have suggested that this disease can be managed with conservative treatment.4–7 However, since 2000, several reports have revealed that this kind of tumor can be malignant with evidence of recurrence.8–10 Although the combination of ultrasonography, computed tomography (CT), magnetic resonance (MR) imaging, and angiography increases the accuracy in diagnosis of hepatic AML, the correct preoperative diagnostic rate of imaging studies has been reported to be less than 50%.6,10–14 Even the postoperative pathologic diagnosis has been easily mistaken as hepatocellular carcinoma (HCC).14,15 Many patients have been treated with surgical resection of the tumor. Therefore, the proper treatment of hepatic AML has remained controversial.
The purpose of this study is to retrospectively review the clinical, imaging, and pathological features of patients with hepatic AML treated at our hospital and to summarize our experience in the diagnosis and treatment of this disease. We also review the literature to highlight the important questions concerning hepatic AML: (1) Is hepatic AML a pure benign tumor? (2) What is the natural course of this tumor? Does the tumor size enlarge frequently during observation? (3) What difficulties exist in preoperative diagnosis with imaging studies and fine needle aspiration biopsy (FNAB)? (4) Is it proper for a hepatitis-carrier patient with hepatic AML to be treated with conservative management? (5) What are the criteria for patients with hepatic AML to be treated with surgical resection or conservative management?
Materials and Methods
The clinical, imaging, and pathological features of 10 patients with hepatic AML treated at the authors’ institute were retrospectively reviewed. The follow-up information was obtained in each case. All tumor tissue was paraffin-embedded for routine hematoxylin and eosin (H&E) staining. Immunohistochemical assays were performed using a three-step indirect peroxidase complex technique with the following antibodies: HMB-45 (DAKO, dilution 1:40), actin (DAKO, dilution 1:50), S-100 (DAKO, dilution 1:800), cytokeratin (Biogenix, dilution 1:80), vimentin (DAKO, dilution 1:50), and c-kit (MBL, dilution 1:200).
Results
Patients and Clinical Data
Ten patients with hepatic angiomyolipoma were diagnosed at National Taiwan University Hospital from July 1995 to June 2004. There was marked female predominance (9/10). The median age was 44 years old with a range from 34 to 64 years. Most patients (60%) presented no symptoms and were detected incidentally by health check-ups or during medical exams for other diseases. Four of 10 patients had symptoms caused by the space-occupying effect of the tumors such as abdominal pain, abdominal fullness, and palpable mass, or other nonspecific symptoms such as fever, general malaise, or body weight loss (Tables 1 and 2). None of them had a history of renal AML or tuberous sclerosis. Two patients were hepatitis B-virus (HBV) carriers. The plasma levels of α-FP and CEA were within normal limits in all patients.
Table 1Clinical Presentation of Hepatic AngiomyolipomaClinical FeatureNo. of PatientsAge34–64 years (median 44 years)Gender (female: male)9:1Symptoms No symptom6 Abdominal pain2 Abdominal fullness2 Palpable mass1 Body weight loss2 Malaise1 Fever2Tumor location Right lobe5 Left lobe4 Caudate1Tumor size (cm) <53 5–101 >106Preoperative diagnosisAngiomyolipoma4(40%) Based on radiological images2 Based on tumor biopsy2Hepatocellular carcinoma3(30%)Angiosarcoma1(10%)Hemangioma1(10%)Metastasis1(10%)Associated liver diseaseHBV carrier2Table 2Clinical Profile of Patients with Hepatic AngiomyolipomaCaseSex/AgeTumor Size (cm)/lobeSymtoms/SignsIncidental FindingTreatmentOutcome/F/U Months1F/3418/RNilH/CAtypical hepatectomyWell/39 mon2F/3410/REpigastralgiaRight lobectomyWell/59 mon3F/3713/LPalpable mass, abdominal fullness, BW loss, feverExtended left lobectomyDead/14 mon recurrent, liver and lung mets4F/4020/REpigastralgiaRight lobectomyWell/109 mon5F/427/RNilH/CFNAB and F/ULost F/U/6mon6F/4611/LAbdominal fullness, malaise, BW loss, feverLeft lateral segmentectomyWell/40 mon7F/4915/RNilExam of appendicitisS56 segmentectomyWell/37 mon8F/513/CNilH/CCaudate lobectomyWell/40 mon9F/532.5/LNilF/U echo due to colon cancer s/pLeft lateral segmentectomyWell/33 mon10M/644/LNilH/CLeft lobectomyWell/32monH/C = health check-up, BW = body weight, F/U = follow-up, FNAB = fine needle aspiration biopsy, mon = month, mets = metastasis, s/p = postoperation
Imaging Studies
Based on the combined imaging studies of abdominal ultrasonography, CT, MR imaging, and angiography, the diagnostic accuracy of hepatic AML in this series was only 20% (Table 1). Other preoperative imaging impressions included hepatocellular carcinoma, angiosarcoma, hemangioma, and metastatic lesions.
Two other cases were diagnosed by fine needle aspiration biopsy (FNAB). The accurate preoperative diagnostic rate was 40% (4/10) after imaging studies and FNAB (Table 1).
Pathologic Study
All 10 patients had a single tumor. Five tumors were in the right lobe of the liver and four were in the left lobe. One tumor was located in the caudate lobe. Most tumor sizes were larger than 5 cm (70%). The median tumor size was 10.5 cm, ranging from 2.5 cm to 20 cm (Tables 1 and 2).
Gross pathology identified all tumors as a well-circumscribed, nonencapsulated tumor masses consisting of soft to elastic tissue. The cut surface in tumors varied from yellow to dark brown.
Histopathologic studies of these 10 tumors showed a picture of hepatic angiomyolipoma composed of myoid and vascular components with a variant content of fatty tissue. Hematopoiesis was noted in two cases. Immunohistochemical studies were performed in all patients except one (case 5). Most tumors were found positive for HMB-45 (10/10), SMA (4/9), S-100 (7/9), Vimentin (6/9), but negative for cytokeratin (0/9). Only three tumors were found positive for c-kit (Table 3).
Table 3Immunohistochemical StudyCaseHMB-45ActinS-100CytokeratinVimentinc-kit1++−++−−+2+++++−++3++−++−+−4++++−−+−5++6+++++−+−7++−++−+−8++−−−+−9++−++−−−10++++−++++: strongly staining, >30% positivity; +: weakly staining, 10∼30% positivity; − no staining, or <10% positivity
Treatment and Follow-up
One patient (case 5) was confirmed with AML through fine needle aspiration biopsy. Nonoperative management with close follow-up was performed. However, this patient was lost after 6 months of follow-up. The other nine patients underwent hepatectomy with tumor resection. These nine patients, except for one patient (case 3), had no postoperative complications or disease recurrence, and were regularly followed up at our outpatient department, follow-up ranging from 32 to 109 months (Table 2). The very unusual patient (case 3) was a 37-year-old woman with a 13 × 9 × 9 cm, large tumor at the left lobe of the liver, receiving extended left lobectomy (Fig. 1a,b). Pathology revealed a picture of hepatic AML (Fig. 2a,b).
Figure 1A 37-year-old woman (case 3) presented with fever and palpable abdominal mass. (a) The axial view of contrast-enhanced CT scans on portal venous phase shows a huge hepatic tumor at the left hepatic lobe with heterogeneous enhancement. Notice the engorged vessels within the tumor are vividly identified (arrow). (b) The MR coronal Tru FISP, fast imaging with steady-state precession. (TR/TE/FA = 4.3/2.1/72°) shows engorged vessels in the tumor. The right portal vein (arrow) is displaced by the tumor. (c) After 6 months of extended left lobectomy, the abdominal ultrasonography reveals a huge recurrent tumor (arrows) in the previous location of left hepatic lobe, and numerous smaller tumors in the right lobe. (d) Celiac angiography also demonstrates the recurrent huge tumor and other multiple smaller ones in the right lobe of liver. Note the early drainage vein (arrow).Figure 2Microscopic appearance of the hepatic angiomyolipoma in case 3. (a) The primary tumor is composed of polygonal to spindle cells arranged in solid sheets or trabecular pattern with endothelial lining. Some of the tumor cells have eosinophilic cytoplasm, and some have large fat vacuoles. Some of the nuclei are bizarre, and some have large eosinophilic nucleoli (H&E stain, original magnification ×100). (b) The tumor cells are strongly immunoreactive for HMB-45 (original magnification ×100). Recurrent tumor was noted 6 months later, and the patient received fine needle aspiration biopsy. (c) Microscopically, it shows tumor cells with clear to ample eosinophilic cytoplasm arranged in trabecular pattern (H&E stain, original magnification ×40). (d) Immunohistochemical staining shows the tumor cells are also positive for HMB-45 (original magnification ×200).
Unfortunately, 6 months later, ultrasonography showed recurrent hepatic lesions at the right lobe of the liver (Fig. 1c). MRI also confirmed a large tumor in the caudate lobe and numerous smaller nodules in the right lobe of the liver. Angiography also revealed multiple tumor stains (Fig. 1d). Fine needle aspiration biopsy was performed. The biopsy specimen was immunoreactive to HMB-45 antibody (Fig. 2c,d). The clinical and histologic picture demonstrated recurrent malignant hepatic angiomyolipoma. At the 11th postoperative month, chest CT scans revealed multiple metastatic nodules. Three months later, the woman died due to hepatic failure and renal failure.
Discussion
In the past, hepatic AML has been considered as a “benign” mesenchymal tumor. However, in 2000, Dalle reported the first case of malignant hepatic AML with vascular invasion and recurrence with multiple liver metastases and suspected portal vein thrombosis 5 months after primary tumor resection.8 Another two cases have been reported with hepatic recurrence after operation. One case was a 16-year-old girl with hepatic AML, receiving left lobectomy, with late recurrence noted 6 years after operation.9 The other one was in the Flemming’s report. Recurrent hepatic tumors were noted 3 years after operation.10 Flemming also suggested that a proliferation index exceeding 3% and multicentric growth indicate a propensity for recurrence.
In this study, we reported a 37-year-old woman with left hepatic AML. A recurrent hepatic mass was noted 6 months after tumor resection, and multiple lung metastases were noted later. The patient died 14 months after diagnosis. To our knowledge, this case is the fourth reported case of recurrence in the literature, and the tumor in this case behaved as the most malignant one.8–10 Therefore, hepatic AML should not be considered as an entirely benign tumor; at least, it has malignant transformation potential. Accordingly, conservative treatment should be performed carefully, especially for patients with poor compliance, who are unable to undergo a strict follow-up regimen.
There were few reports concerning the growth velocity of hepatic AML in long-term follow-up. In one retrospective study of 26 patients, there were six patients who were followed up for more than 1 year and finally decided to receive operation because of the enlargement of the lesions. In that study, the tumor size of one patient increased from 4 to 10 cm during the 5-year follow-up. Another patient had a tumor increasing from 1.5 to 5 cm in 13 years follow-up.13 In another case report of a 38-year-old patient, the tumor size enlarged from 8 to 14.4 cm over a 3-year follow-up period.16 Irie also reported that a 40-year-old woman had hepatic AML with tumor size increasing in size from 4 cm to 7 cm during a 14-month follow-up period.16
Although hepatic AML seems slow-growing, the probability of tumor enlargement and hence an induced mass-compression effect is not uncommon in the long-term follow-up period. In the present series, the median age of patients was 44 years old, and 70% of patients were below 50 years. If all of these patients had received nonoperative management, the mass effect of tumor enlargement might have been presented during a long-term follow-up period, especially in younger patient groups with longer remaining years of life. Moreover, the difficulties and complications of operation at later years would increase when the tumor enlarges, especially for those patients with an original larger tumor (>5 cm).
Similar to the patients presented in this series, most patients with hepatic AML are not symptomatic.12–14 Usually, these patients are diagnosed during health check-ups. Most symptoms are mass-compression effects including upper abdominal pain, abdominal fullness, and palpable mass. There are also some vague symptoms such as body weight loss, general malaise, and fever. In one review article with a collection of 52 patients, the incidence of symptoms or signs dramatically increased when tumor size was larger than 5 cm.11 Twenty-one percent (4/19) of patients with a tumor smaller than 5 cm present symptoms/signs; however, the incidence increases to 64% (7/11) when tumor size is between 5 and 10 cm. The incidence increases to 89% when tumor size is larger than 10 cm. In our series, 40% (4/10) of patients were symptomatic, and all four of these patients had a tumor larger than 10 cm. Accordingly, we suggested that patients with tumor larger than 5 cm should receive tumor resection, because most patients in this group were predisposed toward being symptomatic.
The typical findings in imaging studies of hepatic AML are as follows: (1) heterogeneously hyperechoic mass in US, (2) heterogeneously low density with low attenuation value (less than −20 HU) in plain CT, (3) high intensity on T1 and T2 weighted MRI, and (4) hypervascularity and tumor stain on angiography.12 Although a combination of US, CT, MRI, and angiography is able to increase the accuracy in preoperative diagnosis, hepatic AML usually shows various patterns in imaging studies. The differences in imaging studies occur because the relative proportions of vessels, muscles, and fatty tissue vary widely from one tumor to another. Consequently, hepatic AML is sometimes difficult to diagnose based on imaging studies.17 Therefore, fine needle aspiration biopsy has been reported to be useful in the preoperative diagnosis of this tumor.4,5,17,18
However, more attention should be paid to the tumor’s various morphologic appearances when minute samples are interpreted. With the combined tools of imaging studies and FNAB, the preoperative diagnostic accuracy has been smaller than 32% (ranging from 0 to 32%) in larger series.6,10–15 In a collaborative study reported by Tsui, including 30 cases from nine international hepatology centers, 50% were primarily misdiagnosed as carcinoma or sarcoma, either by imaging studies or by needle biopsy.15 In Flemming’s series, only one preoperative case was diagnosed correctly.10 In the present series, only four preoperative cases (40%) were correctly diagnosed by combined imaging studies and FNAB.
Definite pathologic diagnosis of this tumor is usually made by identification of the three different components of smooth muscle cells, adipose tissue, and blood vessels. HMB-45 positive staining of myoid cells has been used as a pathologic characteristic of hepatic AML.3,19 Because of the rarity and pleomorphism of the histological features of hepatic AML, histologic diagnosis may be difficult, especially with needle biopsy. Many features in AML can mislead the unwary pathologist to a diagnosis of hepatocellular carcinoma: polygonal cells in trabecular arrangement, peliosis, nuclear pleomorphism, prominent eosinophilic nucleoli, deficient reticulin framework, presence of glycogen, eosinophilic globules, and tumor necrosis.14 In Zhong’s series of 2000, none of the 14 cases were correctly diagnosed before operation. Furthermore, five cases were misdiagnosed as hepatocellular carcinoma or sarcoma by pathologists, even after operation. Therefore, we should be cautious when using FNAB as a diagnostic tool.
In an endemic area of hepatocellular carcinoma such as Taiwan,20 conservative management is risky because cases of fat-rich minute hepatocellular carcinoma will make the differential diagnosis more difficult. Furthermore, Chang reported one case with hepatic AML and concomitant hepatocellular carcinoma.21 In this series, two patients were carriers of hepatitis B virus with a high risk for hepatoma formation. Not only would these hepatitis-carrier patients bear more risk, but physicians would also bear more risk and psychological pressure during a long-term follow-up period if conservative management were adopted.
Because of the small patient number, we could not get definitely conclusive management suggestions solely from the results of this retrospective study. But a combination of our experience and a review of the literature, we suggest all symptomatic patients should receive surgical resection for hepatic angiomyolipoma. Conservative management with close follow-up is suggested in patients with asymptomatic tumors and meet the following criteria: (1) tumor size smaller than 5 cm, (2) angiomyolipoma proved through fine needle aspiration biopsy, (3) patients with good compliance, and (4) not a hepatitis-virus carrier.

hepatic angiomyolipoma
angiomyolipoma
liver tumor


Competition and coexistence of sulfate-reducing bacteria, acetogens and methanogens in a lab-scale anaerobic bioreactor as affected by changing substrate to sulfate ratio

The microbial population structure and function of natural anaerobic communities maintained in lab-scale continuously stirred tank reactors at different lactate to sulfate ratios and in the absence of sulfate were analyzed using an integrated approach of molecular techniques and chemical analysis. The population structure, determined by denaturing gradient gel electrophoresis and by the use of oligonucleotide probes, was linked to the functional changes in the reactors. At the influent lactate to sulfate molar ratio of 0.35 mol mol−1, i.e., electron donor limitation, lactate oxidation was mainly carried out by incompletely oxidizing sulfate-reducing bacteria, which formed 80–85% of the total bacterial population. Desulfomicrobium- and Desulfovibrio-like species were the most abundant sulfate-reducing bacteria. Acetogens and methanogenic Archaea were mostly outcompeted, although less than 2% of an acetogenic population could still be observed at this limiting concentration of lactate. In the near absence of sulfate (i.e., at very high lactate/sulfate ratio), acetogens and methanogenic Archaea were the dominant microbial communities. Acetogenic bacteria represented by Dendrosporobacter quercicolus-like species formed more than 70% of the population, while methanogenic bacteria related to uncultured Archaea comprising about 10–15% of the microbial community. At an influent lactate to sulfate molar ratio of 2 mol mol−1, i.e., under sulfate-limiting conditions, a different metabolic route was followed by the mixed anaerobic community. Apparently, lactate was fermented to acetate and propionate, while the majority of sulfidogenesis and methanogenesis were dependent on these fermentation products. This was consistent with the presence of significant levels (40–45% of total bacteria) of D. quercicolus-like heteroacetogens and a corresponding increase of propionate-oxidizing Desulfobulbus-like sulfate-reducing bacteria (20% of the total bacteria). Methanogenic Archaea accounted for 10% of the total microbial community.
Introduction
The anaerobic digestion of organic material is accomplished by the concerted action of various trophic groups of bacteria (Schink et al. 1992). The soluble organic compounds are degraded to CH4, CO2, and H2S via a syntropic interaction of fermentative and acetogenic bacteria with methanogens or sulfate-reducing bacteria (SRB). The electrons for CO2 reduction to methane or acetate, or sulfate reduction to sulfide are derived from the fermentative metabolism. The fermentative metabolism may include homoacetogenic or heteroacetogenic microorganisms depending upon whether acetic acid is the only major metabolic product (Drake 1994; Thauer et al. 1977) or other organic acids, such as propionic acid, in addition to acetic acid are produced (Seeliger et al. 2002). The products of fermentative acetogenesis and CO2-dependent homoacetogenesis can serve as substrates for acetate- and propionate-oxidizing SRB or acetoclastic methanogens depending upon the presence or absence of sulfate as electron acceptor. SRB are believed to outcompete methanogens in the presence of nonlimiting sulfate concentrations because they compete better for common substrates. This is explained on the basis of their kinetic properties (Ks and μmax) and given the favourable thermodynamic conditions (Ward and Winfrey 1985; Widdel 1988). In contrast, methanogens tend to dominate in low-sulfate environments (Stams 1994).
In most anaerobic environments, hydrogen is present as an intermediate for which SRB, hydrogenotrophic methanogens and homoacetogens will compete. Under standard conditions, sulfate reduction and methanogenesis are thermodynamically more favourable than homoacetogenesis. However, due to the interspecies transfer of fermentation products, such as hydrogen, the real thermodynamic values may be far from standard conditions and even less to reverse reactions. Studies with the sludge from the bioreactors have indicated that hydrogen is mainly consumed by SRB when H2 is limiting and sufficient sulfate is present (Isa et al. 1986). However, when H2/CO2 was added as the sole substrate, a coexistence of heterotrophic SRB and homoacetogens was reported by Weijma et al. (2002), suggesting that when SRB are dependent for growth on homoacetogens, it is likely that the growth kinetics of homoacetogens determines the competition (Stams et al. 2005).
The composition of the influent chemical oxygen demand (COD; electron donor) to  ratio tends to have a significant effect on the competition for electron flow between different microbial communities (McCartney and Oleszkiewics 1993). The theoretical  mass ratio needed to achieve complete removal of organic matter is 0.67, assuming that eight electrons are transferred per molecule of sulfate (Lens et al. 1998). In general, low and high  mass ratios should favor sulfidogenesis and methanogenesis, respectively. However, values reported in the literature vary considerably (Annachhatre and Suktrakoolvait 2001; Choi and Rim 1991; Mizuno et al. 1994). Although the effect on the major processes like sulfidogenesis and methanogenesis on the whole has been well studied (McCartney and Oleszkiewicz 1993), our understanding of its influence on the diversity and dynamics of the microbial communities is still limited. This is partially due to the limitations of the traditional microbiological techniques, including pure culture isolations, most probable number estimation, selective enrichments, etc. Fortunately, molecular techniques, like denaturing gradient gel electrophoresis (DGGE; Muyzer et al. 1993) and fluorescence in situ hybridization (FISH; Amann et al. 1995), have provided alternative approaches to overcome the limitations associated with culture-based analyses.
DGGE profiling of 16S rRNA gene fragments amplified from both genomic DNA and reverse-transcribed RNA, i.e., cDNA not only determines the presence and distribution but also the actively growing members in the mixed microbial communities (Dar et al. 2007a). FISH, on the other hand, allows for the identification and quantification at the level of populations and even single cells (Amann et al. 1995).
In the present study, the structure and function of anaerobic communities maintained in lab-scale continuously stirred tank reactors (CSTR) under sulfidogenic and nonsulfidogenic conditions was investigated. An integrated approach of molecular techniques including DGGE and FISH together with chemical analysis was used to link the microbial population dynamics with changes in the lactate (electron donor) concentration and sulfate availability. The focus of our study was the competition and interaction between the dominant microbial communities of SRB, acetogens and methane-producing Archaea.
Materials and methods
Reactor operation
The schematic representation of the experimental design is given in Fig. 1. The start-up reactor (R-0) was inoculated with sludge from a methanogenic wastewater treatment plant treating lactate-containing wastewater. The reactor was operated in a continuous mode for about five to six volume changes at a dilution rate of 0.02 h−1 with lactate as the only electron donor and maintained under excess of sulfate. The lactate (12 electron) to sulfate (8 electron) molar ratio (r) was 0.65 mol mol−1. The culture in the reactor R-0 was used as an inoculum for the reactors, R1 and R2. The reactors R1 and R2 were operated at 30°C with a working volume of 2 l and at two different concentrations of lactate, 3.5 and 20 mM, respectively. Sulfate was fed to the reactors at a concentration of 10.32 mM, i.e., at ratios far below and far above the theoretical ratio of 0.67. Nonsulfidogenic conditions were applied to reactor R2 by further reducing influent sulfate feed and reducing the lactate concentration to 6.7 mM (r = 20.9). The composition of influent mineral medium was as follows (concentration in mM): KH2PO4, 3.6; Na2SO4, 10 or 0; NH4Cl, 6; MgSO4·7H2O, 0.3; CaCl2·2H2O, 0.4; FeSO4·7H2O, 0.02; a stock trace element solution, 1 ml/l; a stock selenite–tungstate solution, 1 ml/l; and a stock vitamin solution, 1 ml/l. The stock trace element solution contained (mg/l): H3BO3, 30; MnCl2·4H2O, 100; CoCl2·6H2O, 190; NiCl2·6H2O, 24; CuCl2·2H2O, 2; ZnCl2, 140; Na2MoO4·2H2O, 36. The stock selenite–tungstate solution contained (mg/l): Na2SeO3·5H2O, 6 and Na2WO4·2H2O, 8. The stock vitamin solution was composed of (mg/l): biotin, 10; vitamin B12, p-aminobenzoic acid, and calcium D (+) pantothenate, 50; thiamine, pyridoxine–HCl, and nicotinic acid, 100. The medium was autoclaved, and the dissolved oxygen removed from the hot media by sparging it with oxygen-free nitrogen. The medium was maintained in an oxygen-free atmosphere by continuously flushing it with nitrogen. The pH in the reactors was maintained at 7.5 by automatic titration with 1 M NaOH and 1 M HCl. An automatic level controller maintained the working volume of 2 l inside the reactor vessels. The cultures were continuously sparged with oxygen-free nitrogen to flush the gaseous H2S. Sulfide precipitated as ZnS after letting the gaseous H2S through a zinc acetate trap connected to the gas exhaust of the reactor. Cultures were run at D = 0.02 h−1 for at least five to six volume changes (one volume change is 50 h) and until chemical analysis of effluent showed constancy over at least two consecutive days.
Fig. 1Schematic representation of the experiment. Reactor R-0 was the start-up reactor. The culture in R-0 was used as an inoculum for the reactors R1 and R2a. The cultures were run at a dilution rate of 0.02 h−1 for five to six volume changes with each volume change of 50 h
Chemical analysis
Acetate and other volatile fatty acids were analyzed either by gas chromatography (GC) or by high-performance liquid chromatography (HPLC). Sulfate was analyzed by ion chromatography as described previously (Scholten and Stams 1995). Sulfide was measured quantitatively by a colorimetric assay (Cline 1969). As sulfide was stripped from the culture liquid with nitrogen to reduce sulfide toxicity, the sulfide produced was calculated from the sulfate consumed. Hence, the given sulfide values indicate minimum values.
Nucleic acid extraction, reverse transcription of RNA, and PCR amplification
Nucleic acid extraction was carried out as described previously (Dar et al. 2007b). One microliter (80–100 ng) of RNA template was used to carry out the reverse transcription (RT) of the isolated RNA into cDNA. Reverse transcription was performed using the iScript cDNA Synthesis Kit (BioRad, California, USA) according to the manufacturer’s protocol. Amplification of the 16S rRNA gene fragment was performed using the primer pair 341F-GC and 907R and the protocol as described previously (Muyzer et al. 1995). Nested amplification with group-specific primers for SRB was also performed as described previously (Dar et al. 2005). We used 1 μl of genomic DNA and 2 μl of cDNA as template for the amplification reactions. The quality of the PCR products was examined on 1% (w/v) agarose gel, and the yield was quantified by absorption spectrophotometry using the Nanodrop ND-1000 TM (NanoDrop Technologies, Delaware, USA).
DGGE of 16S rRNA gene fragments and phylogenetic analysis
DGGE was performed as described by Schäfer and Muyzer (2001) using the D-Code system (BioRad Laboratories, California, USA). After electrophoresis, the gels were incubated for 30 min in a solution containing ethidium bromide (0.5 μg/ml), rinsed for 20 min in Milli-Q water, and photographed using a BioRad GelDoc station (BioRad, California, USA). Individual bands were excised, resuspended in 20 μl of Milli-Q water, and stored overnight at 4°C. A volume of 3 to 5 μl of the supernatant was used for reamplification with the original primer sets. The reamplified PCR products were run again on a denaturing gradient gel to check their purity. Before sequencing, the PCR products were purified using the Qiaquick PCR purification kit (QIAGEN, Hilden, Germany).
The obtained 16S rRNA gene sequences were first compared to the sequences stored in publicly accessible database using the NCBI BLAST search tool (http://www.ncbi.nlm.nih.gov/BLAST; McGinnis and Madden 2004). Subsequently, the sequences were imported into the ARB software program (Ludwig et al. 2004) and aligned using the automatic aligner function. The alignment was further corrected manually, and an optimized tree was calculated using the neighbor-joining algorithm with Felsenstein correction.
Design of oligonucleotide probes
Specific oligonucleotide probes for the 16S rRNA of the dominant anaerobic communities in the reactors were designed using the probe design tool of the ARB software package (Ludwig et al. 2004) as described previously (Dar et al. 2007a). The oligonucleotides used for in situ hybridisation are given in Table 1.
Table 1Oligonucleotides used in this studyProbe nameTarget organismProbe sequence (5′–3′)ReferenceEUB338IMost bacteriaGCT GCC TCC CGT AGG AGTAmann et al. 1990EUB338IIPhylum PlanctomycetesGCA GCC ACC CGT AGG TGTDaims et al. 1999EUB338IIIPhylum VerrucomicrobiaGCT GCC ACC CGT AGG TGTDaims et al. 1999ARCH915ArchaeaGTG CTC CCC CGC CAA TTCStahl and Amann 1991SPS770Genus SporomusaATC CCG TTC ACT CCC CTGThis studySRB385Most DeltaproteobacteriaCGC GTC GCT GCG TCA GGAmann et al. 1990SRB385DbSome DeltaproteobacteriaCGG CGT TGC TGC GTC AGGRabus et al. 1996DSR660Genus DesulfobulbusGAA TTC CAC TTT CCC CTC TGDevereux et al. 1992DSM1265Genus DesulfomicrobiumAGA TTC GCT CGA CCT CGCThis studyDSV139Desulfovibrio sp. strain L7CGC TGT TAT CCC GAT CACDar et al. 2007aDSCOC814Desulfococcus groupACC TAG TGA TCA ACG TTTDevereux et al. 1992
Fluorescence in situ hybridization
Fixed cell samples from the reactors were immobilized on Teflon-coated multiwell microscopic slides as described previously (Dar et al. 2007b). Hybridization was carried out according to the protocol as described previously (Manz et al. 1992) using a formamide concentration of 35% (v/v). Quantification of the hybridized cells was performed as described previously (Neef et al. 1996). The hybridized cells were analyzed by two independent observers for determining the fraction of positive signal from each probe relative to the signal visualized with general probes for bacteria (EUB338 I, II, and III), sulfate-reducing bacteria (SRB385 and SRB385Db), or with the general DNA stain DAPI (4′,6′-diamidino-2-phenylindole). In addition, a general probe specific for members of the domain Archaea (ARC915) was used. The hybridization experiments were done in duplicate, using different fluorochromes for each probe. Different microscopic fields on each slide were analyzed to confirm the results.
Hybridization stringencies of the newly designed probes were determined by performing hybridizations with increasing formamide concentrations as described previously (Manz et al. 1992) using target organism(s) and nontarget organism displaying three mismatches within the target region.
Sequence accession numbers
The sequences determined in this study were submitted to GenBank under accession numbers EU276620–EU276626.
Results
Microbial community dynamics at different lactate/sulfate ratio
Figure 2 summarizes the effluent composition in the bioreactors, after a minimum selection period of five to six volume changes, as a function of the different lactate to sulfate ratios imposed. The start-up reactor R-0 had a lactate/sulfate ratio (r = 0.65 mol mol−1) theoretically sufficient to allow full lactate oxidation to CO2 with sulfate (also see Table 2). The results for R1 with excess sulfate (r = 0.34 mo/mol) indicate that after five to six volume changes only sulfate, sulfide, and acetate, and no other organic acids were detected in the effluent of the bioreactor (Fig. 2). Nearly all lactate (3.5 mM) had been converted to acetate (3.2 mM) with the equivalent conversion of sulfate to sulfide. These measurements indicate partial oxidation of lactate to acetate, coupled to sulfate reduction was an important reaction in R1 (reaction 2, Table 2). At a high lactate to sulfate ratio (r = 1.94 mol mol−1) in the reactor R2a, propionate was detected in the effluent besides acetate (Fig. 2), demonstrating that lactate was partially fermented to a propionate and acetate mixture (reaction 3, Table 2). In this sulfate-limited reactor, nearly all sulfate had been converted to sulfide. At a very low sulfate concentration (r = 20.9), lactate fermentation to acetate and propionate was observed as a dominant reaction (reactor R2b). The shift in the microbial population structure due to different lactate to sulfate ratios after five to six volume changes was recorded by DGGE profiling of 16S rRNA gene fragments, which were amplified either from genomic DNA or from reverse-transcribed RNA, i.e., cDNA.
Fig. 2Effluent concentrations of sulfide, sulfate, acetate, and propionate in reactors R1, R2a, and R2b. The sulfide data are minimum numbers due to N2 strippingTable 2Stoichiometry of anaerobic degradation reactions relevant to this studyNo.ReactionΔG01 kJ reaction−11−128.52−80.83−55.74−37.75−85.46−31.1
The comparative analysis of the profiles based on the DNA and RNA of the same sample reflect not only the presence but also the actively growing populations. Figure 3 illustrates the results of the bacterial 16S rRNA–DGGE reflecting the shift in the predominant populations with the changing lactate to sulfate ratios. For the three lactate to sulfate ratios used (i.e., r = 0.34, 1.94, and 20.9), the DGGE profiles based on 16S rRNA were not identical to the 16S rDNA-based profiles of the same sample. The phylogenetic affiliation of 16S rRNA gene sequences is presented in Fig. 4. A neighbor-joining tree was generated using the sequences of the DNA fragments excised from the denaturing gel. Band B3, representing the most persistent species of all the microbial communities in the DNA based profiles, showed high sequence similarity to the Sporomusa–Pectinatus–Selenomonas group with the closest relative being Dendrosporobacter quercicolus. The DGGE profile of the samples taken from R2a to R2b showed an increase in the intensity of fragment B3. Fragment B5 was abundant in reactors R1 and R2a but with varying degrees of intensity. The fragment was not only persistently present but also belonged to an actively growing population as suggested by its simultaneous presence in the RNA-based profiles. However, the fragment was not present in the DGGE profile when the sulfate feed to the reactor R2b was brought down (r = 20.9). Comparative sequence analysis showed that band B5 clustered with the genus Desulfovibrio with Desulfovibrio mexicoense as the closest relative among the cultured representatives. Fragment B6, which was also significantly present, especially in the RNA-based profiles of R1 and R2a, was related to the members of genus Desulfobulbus with the closest relative being Desulfobulbus rhabdoformis. Band B4 was observed only in R1 maintained at a limiting lactate concentration (r = 0.34 mol mol−1). The fragment was found closely related to Desulfomicrobium apsheronum. Two other fragments excised, band B1 and band B2, although present, did not form a significant proportion of the active microbial communities in R1 and R2 as indicated by the RNA-based profiles. The two fragments were affiliated to an uncultured group of Cytophaga and Spirochetes, respectively. Nested amplification with group-specific primers (results not shown) resulted in the identification of an acetate-utilizing SRB represented by band B7. The sequence of the fragment was found closely related to Desulfococcus biacutus.
Fig. 3DGGE analysis of 16S rRNA gene fragments using DNA and RNA samples from reactors R-0, R1, R2a, and R2b. Lanes 1 (DNA) and 2 (RNA) sample from reactor R-0 (influent lactate 6.7 mM); lanes 3 (DNA) and 4 (RNA) sample from reactor R1 (influent lactate 3.5 mM); lanes 5 (DNA) and 6 (RNA) from reactor R2a (influent lactate 20 mM); lanes 7 (DNA) and 8 (RNA) from reactor R2b (in the near absence of sulfate). Bands indicated with a dot were excised and sequencedFig. 4Phylogenetic tree based on 16S rRNA gene sequences obtained from the DGGE bands (see Fig. 3). Sequences determined in this study are in boldface. Black dots on the nodes indicate bootstrap values of 90% and higher (1,000 replicates). The scale bar indicates 10% sequence difference
The archaeal 16S rRNA gene-targeted DGGE indicated that the diversity was limited to a single species (results not shown) and it was found at all lactate to sulfate ratios except the lowest ratio (r = 0.34) with 3.5 mM lactate. Sequence analysis of the excised fragment indicated its close relatedness to a group of uncultured Archaea distantly related to Methanosaeta sp.
Microbial community dynamics using FISH
The FISH results were obtained using oligonucleotide probes that targeted dominant microbial communities present under different substrate concentrations. The results are presented as relative abundances of populations targeted by specific probes and are expressed as the percentage of the total positive signal obtained by general probes for bacteria (EUB338 I, II, and III) or with the general DNA stain DAPI (4′,6′-diamidino-2-phenylindole). In addition, the general probe specific to Archaea (ARC915) gave a measure of Archaea. The percentage abundance of the specific probes relative to the general probes is summarized in Fig. 5a and b. In general, probe EUB338 stained more than 90% of all the cells in the samples. Probe SPS770 specifically targeted the species that was closely related to D. quercicolus. At the influent lactate concentration of 3.5 mM (reactor R1), the relative abundance of this organism was <2% of the total bacterial population (Fig. 5a and 6b). When compared to the start-up reactor R-0, the relative abundance of the cells targeted by probe SPS770 was 8–10% of the total bacterial cells (Fig. 5a and 6a). The relative percentage of cells that hybridized with this probe was significantly higher (40–45% of the total bacterial cells), in samples taken from reactor R2a (lactate excess, 20 mM; Fig. 5a and 6c) and increasing even higher to more than 70% (Fig. 5a and 6d), when the sulfate feed to reactor R2b was minimized. Probes DSR660, DSV139, and DSM1265 were used to target different sulfate-reducing communities (Table 1) that were identified in the reactor samples using DGGE. Desulfomicrobium-like SRB targeted by probe DSM1265 were present in high proportions at excess sulfate (reactor R1, r = 0.34 mol mol−1). The percentage abundance relative to the total bacteria was more than 65% compared to 15–20% in the inoculum (R-0; Fig. 5b). The numbers were about 5–8% at limiting sulfate concentrations in reactor R2a (r = 1.94) and decreased further to undetectable levels in the virtual absence of sulfate in reactor R2b (r = 20.9). Desulfovibrio-like SRB targeted by probe DSV139 were the most resilient of all the SRB. Their numbers did not change significantly, fluctuating between 10% and 7% of the total bacteria in the two reactors R1 and R2-a, respectively (Fig. 5b). They were reduced to undetectable levels when the sulfate supply to reactor R2-b was virtually stopped.
Fig. 5a Relative abundance of Archaea, sulfate-reducing bacteria, and acetogenic bacteria. b Relative abundance of Desulfomicrobium-, Desulfovibrio-, Desulfobulbus-, and Dendrosporobacter-like bacteria. The percentage abundance is relative to the signal obtained with probe EUBmixFig. 6Whole-cell hybridization of samples from reactors R-0, R1, R2a, and R2b probe EUBmix labeled with Cy5 (blue), probe SRB385 and 385Db labeled with Fluos (green), and probe SPS770 labeled with Cy3 (red). a Reactor R-0 (influent lactate 6.7 mM); b reactor R1 (influent lactate 3.5 mM); c reactor R2a (influent lactate 20 mM); d reactor R2b (in the near absence of sulfate)
The relative abundance of Desulfobulbus-like SRB targeted by probe DSR660 showed the most significant increase among all SRB detected, responding to an increase in lactate concentration combined with sulfate limitation. The percentage abundance increased from about 10–12% in the inoculum (reactor R-0; Fig. 5b) to about 20% of the total bacteria in reactor R2a maintained at limiting sulfate concentration (r = 1.94). In reactor R1 (i.e., at sulfate excess conditions), the Desulfobulbus-like SRB were <5% of the total bacteria (Fig. 5b).
The change to a low sulfate concentration in the reactor resulted in a considerable increase in the methanogenic population. FISH with probe ARCH915 indicated an increase of the methanogenic population from 5% in the inoculum (R-0) to more than 10–15% under limiting sulfate (R2-a) and nonsulfidogenic (R2-b) conditions (Fig. 5a). The relative abundance of the methanogenic population decreased to undetectable levels after the shift from R-0 to the lactate-limited reactor R1.
Discussion
This study evaluates the shift in microbial structure in lab-scale CSTR reactors as affected by changing lactate to sulfate ratios using a combined approach of molecular methods (PCR–DGGE and FISH) and chemical analysis. The combination of different methods allowed the establishment of a link between the population structure and function of the anaerobic communities in the reactors under sulfidogenic and nonsulfidogenic conditions.
PCR–DGGE profiles produced using DNA templates may not reflect the dynamic changes in community structure. Metabolically active cells have a much higher ratio of rRNA to DNA than dormant cells (Weller and Ward 1989). Therefore, the use of rRNA templates may emphasize the actively growing populations in the community as opposed to those that are relatively inactive but persistent in the community. Because PCR-based approaches for the analysis of microbial diversity in mixed populations can be influenced by several constraints (von Wintzingerode et al. 1997), our results based on PCR–DGGE do not necessarily reflect the abundance of target sequences in the reactor samples. We, therefore, tried to confirm the relevance of the sequence data by whole-cell hybridization using fluorescently labeled oligonucleotide probes.
Depending on different lactate to sulfate ratios, theoretically, various scenarios involving SRB in the degradation of lactate are possible as shown in Table 2.
Complete oxidation of lactate to CO2 and sulfide by SRB (reaction 1, Table 2).Incomplete oxidation of lactate to acetate by SRB (reaction 2, Table 2).Fermentative degradation of lactate to propionate and acetate and the utilization of fermentation products by SRB and acetoclastic methanogens (reactions 3, 4, 5, and 6, Table 2).In the absence of sulfate the fermentative degradation of lactate coupled to acetoclastic methanogens (reactions 3 and 6, Table 2).
The net metabolic pathway of the degradation of lactate by a mixed culture inoculum used in this study suggests its dependence upon the relative quantities of lactate and sulfate. Theoretically, the complete oxidation of lactate to inorganic carbon requires a minimum lactate to sulfate ratio of 0.67 mol mol−1 (reaction 1, Table 2) and if the ratio is below 0.67, additional electron donor is required for complete sulfate reduction. At lower molar ratio of lactate to sulfate (0.34 mol mol−1, reactor R1), only sulfate, sulfide, and acetate were detected in the effluent. These results directly show the quantitative oxidation of lactate to acetate coupled to sulfate reduction as a dominant reaction (reaction 2, Table 2). This is consistent with high percentage abundance of Desulfomicrobium- and Desulfovibrio-like SRB (known, incompletely oxidizing, SRB capable of performing reaction 2, Table 2; Figs. 5a and 6b; Widdel 1988). Complete lactate oxidation to carbon dioxide with sulfate as electron acceptor would be energetically favorable (reaction 1, Table 2). However, the kinetics of this complete oxidation may not enable efficient competition with the incomplete lactate oxidizers. This is evident from the observation that in batch culture in the presence of excess sulfate, incompletely oxidizing SRB outcompete completely oxidizing species due to their superior maximum specific growth rate on fermentation intermediates like lactate, ethanol, propionate, etc. (McCartney and Oleszkiewicz 1993; Widdel 1988). The actual competition between partial and complete lactate oxidizing SRBs will be determined by the affinity for lactate (μmax/Ks).
Given the fact that the nested amplification suggested that the numbers of the complete oxidizers were very low in the inoculum, a long period of time would have been required before such SRB would have grown to a significant number. Therefore, we cannot rule out that eventually the complete oxidizers might have become dominant under lactate-limiting conditions in R1.
In the near absence of sulfate, lactate fermentation to acetate and propionate (reaction 3, Table 2) was observed as a dominant reaction in the reactor R2b. This was reflected by a significant increase in the relative abundance of Dendrosporobacter-like species and a near washout of SRB species (Fig. 5a and 6d). D. quercicolus is an obligate anaerobe that clusters with the Sporomusa–Pectinatus–Selenomonas group (Strompl et al. 2000). The members of this group are known to perform acetogenic and propionic acid fermentation (reaction 3, Table 2; Biebl et al. 2000). Major fermentation products of D. quercicolus and other closely-related members of the group are acetate and propionate. Theoretically (reaction 3, Table 2), the fermentation of 6.7 mM of lactate should yield approximately 4 mM of propionate and 2 mM of acetate assuming that 0.5 mM is assimilated into the biomass. However, the acetate concentration in the effluent of the reactor R2b (Fig. 2) indicated a net loss of 15% of the electron donor. The plausible explanation for this loss can be acetoclastic methanogenesis (reaction 6, Table 2) that can be assumed from an increase in the methanogenic population to 15% under nonsulfidogenic conditions (Fig. 5a).
The lactate to sulfate molar ratio in reactor R2a (1.94 mol mol−1) was increased by a factor of three compared to the inoculum, reactor R-0 (0.67 mol mol−1) and a factor of about six compared to reactor R1 (0.34 mol mol−1). The conditions in reactor R2a were changed to sulfate limitation with a substantial increase in influent lactate concentrations. Indeed, the sulfate concentration had dropped to <0.2 mM, showing that the SRBs could no longer compete for the remainder of the substrates. The products in the effluent included propionate and acetate besides sulfide, suggesting that lactate was partially fermented to a propionate and acetate mixture (reaction 3, Table 2). This indicates a change in the net metabolic pathway for the degradation of lactate compared to the reactor R1. This change may be attributed to the lactate fermenting bacteria catalyzing reaction 3 (Table 2). The same is suggested by a significant increase in the population of D. quercicolus-like species, 5 times increase relative to reactor R-0 and even 20–25 times relative to reactor R1 (Fig. 5a and 6c). This is in agreement with the previous studies performed (McCartney and Oleszkiewicz 1993; Mendez et al. 1989; Pichon et al. 1988; Ueki et al. 1986). In all these studies, an increase in the buildup of propionate was observed with increasing organic substrate to sulfate ratio. McCartney and Oleszkiewicz (1993) attributed this change to the net metabolism of lactate, the combined influence of the increased lactate to sulfate ratio, and the time for which the culture was exposed to excess sulfate. This is clearly also the case in our experiments: if a steady state has been reached, the DNA and RNA bands in the DGGE are expected to be similar, if not identical. As demonstrated in Fig. 3, this was not the case. Again, if growth rates would have been only marginally different, a true steady state might have taken several weeks to become established, and hence, we cannot rule out that we have looked at a snapshot during a shift in the population. In this study, the exposure of the original inoculum from the anaerobic wastewater plant to excess sulfate was approximately 13 days during start-up reactor, R-0, before an increase in the lactate to sulfate ratio was imposed in reactor R2a (1.94 mol mol−1). This exposure time was enough to enrich SRB in sufficiently high numbers for the development of substantial sulfidogenesis leading to sulfate depletion. As pointed out above, the consequence was that the culture runs into sulfate limitation and, under this condition, acetogens and methanogens experience no competition for the remaining substrate. It is interesting to note that the buildup of propionate in reactor R2a was concurrent to the increase in D. rhabdoformis-like SRB (Fig. 5b), which are known to degrade propionate (reaction 4, Table 2). This increase in the relative abundance of D. rhabdoformis-like SRB, suggesting a synergistic association between heteroacetogenic bacteria and SRB, is a feasible scenario in the presence of limiting sulfate.
An overall electron balance over the system was established, assuming a biomass yield of 0.07°C-mol C-mol−1. In the experiment, at a lactate to sulfate ratio of 0.34 mol mol−1 (reactor R1), the electron balance was nearly 95%. However, in the other experiments with R2, a net loss of reduction equivalents between 10% and 50% is observed. This may be attributed to methane or H2 formations, which were stripped out with the sulfide. Assuming the dominance of partial lactate oxidation coupled to sulfate reduction in reactor R1 (0.35 mol mol−1) and a partial lactate fermentation to propionate and acetate in reactor R2-a (1.94 mol mol−1), both H2 and acetate are the most plausible precursor for methane formation. However, the observation that relatively high concentrations of acetate were found in the reactor effluent (1–4 mM) and the detection of methanogenic Archaea which clustered with Methanoseata sp. suggests that, given the dilution rate of 0.02 h−1, only a low affinity acetoclastic methanogen catalyzing reaction (reaction 6, Table 2) could be maintained in the system. These results were consistent with the increase in the percentage abundance of methanogenic Archaea to more than 10–15% of the total DAPI-stained cells. Because the conditions in the reactors did not favor the growth of nonmethanogenic Archaea (extreme halophiles, thermoacidophiles, the Archaeoglobales, and thermophiles placed in Crenarchaeota kingdom; Woese 1987), cells targeted by probe ARCH915 can be assumed to be the total methanogenic representation in this environment.
In conclusion, this study points to different metabolic routes being followed by a mixed anaerobic community, even in the mineralization of simple substrates like lactate. We could establish a link in microbial population dynamics to major perturbations caused by changing influent lactate to sulfate ratios. This study demonstrated that at low lactate to sulfate molar ratios in the influent, SRB had a competitive advantage over acetogens and methanogens and that, in the near absence of sulfate, heteroacetogens formed a syntropic association with methanogens. Higher lactate to sulfate ratio resulted in a pathway that had propionate and acetate as products, and the majority of sulfidogenesis and methanogenesis was dependent on the fermentation products. All these results were substantiated by corresponding shifts in relative abundance of the microbial communities present as analyzed by DGGE and FISH.

sulfate-reducing bacteria
acetogens
methanogens
dgge
fish
anaerobic consortia


Experimental models for the autoimmune and inflammatory blistering disease, Bullous pemphigoid

Bullous pemphigoid (BP) is a subepidermal skin blistering disease characterized immunohistologically by dermal-epidermal junction (DEJ) separation, an inflammatory cell infiltrate in the upper dermis, and autoantibodies targeted toward the hemidesmosomal proteins BP230 and BP180. Development of an IgG passive transfer mouse model of BP that reproduces these key features of human BP has demonstrated that subepidermal blistering is initiated by anti-BP180 antibodies and mediated by complement activation, mast cell degranulation, neutrophil infiltration, and proteinase secretion. This model is not compatible with study of human pathogenic antibodies, as the human and murine antigenic epitopes are not cross-reactive. The development of two novel humanized mouse models for the first time has enabled study of disease mechanisms caused by BP autoantibodies, and presents an ideal in vivo system to test novel therapeutic strategies for disease management.
Etiology of bullous pemphigoid
In 1953, Lever [28] described bullous pemphigoid (BP) as a subepidermal blistering disorder primarily seen in the elderly. Lesional/perilesional skin of BP patients exhibits detachment of the basal keratinocytes of the epidermis from the dermis at the level of the lamina lucida [55], resulting in tense, fluid-filled vesicles. BP is both an inflammatory disease and an autoimmune disease, characterized by an inflammatory infiltrate at the site of the dermal–epidermal junction separation and by the deposition of autoantibodies and complement components along the basement membrane zone (BMZ).
A number of inflammatory cells are present in the upper dermis and bullous cavity, including eosinophils (the predominant cell type), neutrophils, lymphocytes, and monocytes/macrophages. Both intact and degranulating eosinophils, neutrophils, and mast cells (MC) are found in the dermis. Local activation of these cells may occur via the multiple inflammatory mediators present in the lesional skin and/or blister fluids, including (a) granular proteins derived from degranulated leukocytes, such as eosinophil cationic protein (ECP), eosinophil major basic protein (MBP), and neutrophil-derived myeloperoxidase (MPO) [1, 4, 8] and (b) chemoattractants and cytokines, such as C5a fragments, histamine, leukotriene B4, interleukin-1, -2, -4, 5, -6, -8, -15, TNF-α, IFN-γ, RANTES, and eotaxin [9, 10, 21, 22, 46, 47, 48, 58, 62]. Additionally, several proteinases are found in BP blister fluid, including plasmin, collagenase, elastase, and 92-kDa gelatinase [2, 14, 24, 27, 44, 45, 52, 57]. These proteolytic enzymes may play a crucial role subepidermal blister formation in BP via their ability to degrade extracellular matrix proteins.
BP patients generate a polyclonal repertoire of autoantibodies that bind to the BMZ and activate complement, as well as circulating autoantibodies [20]. These autoantibodies target two major hemidesmosomal antigens of 230 kD (BP230 or BPAG1) and 180 kD (BP180, BPAG2, or type XVII collagen) [25, 40, 56, 57]. BP230, a component of the hemidesmosomal plaque, is an intracellular protein, while BP180 is a type II transmembrane protein [19, 23, 56]. Like BP230, BP180’s amino-terminal portion localizes to the intracellular hemidesmosomal plaque [15, 18, 19]. Its carboxyl-terminal region extends into the extracellular milieu of the BMZ, making it the preferred target for pathogenic BP autoantibodies. This antigenic extracellular region consists of 15 collagen domains separated from one another by non-collagen sequences. The largest of these non-collagen domains is referred to as NC16A. Epitope mapping studies indicate that BP autoantibodies of IgE and IgG isotypes and IgG1 and IgG4 subclasses recognize multiple epitopes that cluster within BP180 NC16A [3, 11, 16, 26, 63]. Serum levels of these autoantibodies are correlated with disease severity [11, 17, 49].
Most BP patients elicit a cell mediated autoimmune response in addition to the humoral response described. Autoreactive CD4+ T lymphocytes recognize epitopes within the extracellular region of BP180, primarily in the NC16A domain [5, 29]. These T cells express memory cell surface markers and exhibit a Th1/Th2 mixed cytokine profile. These studies suggest that BP is a T and B cell-dependent and antibody-mediated skin autoimmune disease.
Development of murine IgG passive transfer model of BP
The strong correlation between BP disease severity and serum BP180-specific autoantibody levels suggests that BP blister formation is mediated by autoantibodies. Early attempts to demonstrate the pathogenicity of patient autoantibodies via a passive transfer mouse model were unsuccessful because BP autoantibodies that react with an immunodominant and potentially pathogenic epitope in BP180-NC16A fail to cross-react with the murine form of this autoantigen (mBP180 NC14A) [30]. In 1993, Liu et al. [30] devised a strategy to overcome this difficulty and generated rabbit polyclonal antibodies raised against a cloned segment of mBP180 NC14A and passively transferred the purified rabbit anti-mBP180 IgG into neonatal BALB/c mice. The injected animals developed a disease that exhibited the following hallmarks of human BP: (a) clinical skin lesions; (b) in vivo deposition of rabbit IgG and mouse C3 at the basement membrane by direct IF; (c) dermal-epidermal separation and an extensive inflammatory cell infiltration by H&E staining [30]. This infiltrate includes neutrophils, lymphocytes, and monocytes/macrophages, with neutrophils being the predominant cells [7, 30].
Immunopathogenesis of experimental BP in the murine model
Development of an in vivo system to study an experimental BP model has allowed for great progress in defining the etiopathogenesis of disease. Specifically, the roles of pathogenic antibodies, the complement system, inflammatory cells, and proteolytic enzymes have all been elucidated in the context of the murine IgG passive transfer model.
Injection of anti-mBP180 IgG initiated subepidermal blister formation, and the levels of circulating anti-mBP180 antibodies completely determine disease onset and severity [30, 34]. Epitope mapping studies demonstrated that pathogenic anti-BP180 antibodies recognize a 9–12 amino acid stretch within the murine BP180 NC14A region of the antigen [31]. This epitope overlaps the region of the human BP180 NC16A that contains the immunodominant epitopes recognized by human BP autoantibodies, supporting the relevance of this murine system as a model for human disease.
Using mice deficient in different inflammatory cell types, it has been demonstrated that mast cells, macrophages, and neutrophils, but not T and B lymphocytes, play a direct role in subepidermal blistering in experimental BP [7]. Anti-mBP180 autoantibodies trigger skin blistering in wild-type mice and mice deficient in T, B, or both T and B cells, but fail to induce lesions in mice deficient in mast cells, macrophages, or neutrophils. Quantification of disease severity in these mice demonstrated that mast cells and neutrophils play a key role in experimental BP.
Autoantibodies bind to basement membrane antigens and activate complement in human BP. In the rabbit anti-mBP180 IgG-induced BP, complement activation is absolutely required. Both BALB/c mice depleted of complement (by pretreatment with cobra venom) and C5-deficient mice are resistant to experimental BP, and reconstitution of the C5-deficient mice with C5a restores susceptibility [32]. F(ab’)2 fragments generated from the pathogenic anti-mBP180 IgG cannot induce subepidermal blisters in C5-sufficient mice [32]. Mice deficient in complement component C4 (specific for the classical and lectin pathways), but not in factor B (specific for the alternative pathway), are resistant to BP [41]. Wild-type mice depleted of complement component C1q (specific for the classical pathway) fail to develop BP skin lesions when injected with pathogenic IgG [41]. These data suggest that the classical pathway of the complement activation plays a major role in development of disease, namely through the generation of C5a that in turn activates MCs [6].
Mice injected with pathogenic anti-mBP180 antibodies exhibit extensive MC degranulation in the lesional skin, similar to that observed in human BP [6, 59]. MC-deficient mice are resistant to experimental BP, but reconstitution of these mice with MCs restores their susceptibility to disease. MC activation precedes neutrophil infiltration, and either the absence of MCs or the inhibition of MC degranulation prevents neutrophil infiltration and blister formation. However, MC-deficient mice reconstituted locally with neutrophils, or injected locally with neutrophil chemoattractants IL-8 or TNF-α form blisters in response to anti-mBP180 IgG. These results suggest that mast cells release proinflammatory cytokines critical for neutrophil recruitment [6].
Experimental BP requires neutrophil infiltration, and there is a direct correlation between disease severity and number of infiltrating neutrophils [33]. Depletion of neutrophils, or disruption of the events upstream of neutrophil infiltration (complement activation and MC degranulation), renders mice resistant to BP blister formation. However, complement system and MC deficiencies only confer protection from the pathogenic effects of BP180 autoantibodies if neutrophil infiltration is blocked as a consequence; that is, complement activation and MC degranulation are only critical to disease progression in their roles of neutrophil recruitment. This suggests that infiltrating neutrophils are the cells that directly cause tissue injury in the dermal–epidermal junction, leading to BP skin blisters.
Upon activation through molecular interactions between the FcγIIIR on the neutrophil cell surface and the Fc portion of pathogenic anti-mBP180 IgG [61], infiltrating neutrophils secrete proteolytic enzymes known to degrade the extracellular matrix, including neutrophil elastase (NE) and gelatinase B (GB). A deficit of either of these two enzymes blocks blister formation in mice [35, 36]. In vitro, although both GB and NE are capable of degrading the recombinant BP180 protein, only NE produces DEJ separation when incubated with skin sections [36, 52]. In vivo, the degradation of BP180 depends on NE activity. Taken together, these findings suggest that GB acts upstream of NE. Specifically, GB proteolytically inactivates the physiological inhibitor of NE (α1-proteinase inhibitor), which allows for unmitigated NE cleavage of extracellular matrix proteins (including BP180), resulting in DEJ separation [37].
Development of the humanized murine passive transfer model
While the rabbit anti-mBP180 IgG passive transfer model has provided invaluable insight to the key steps in BP disease development, it does not allow for experimentation with BP autoantibodies isolated from human clinical samples. A second animal model, in which rabbit antibodies directed against the extracellular domain of hamster BP180 (NC16A region) are transferred into neonatal hamsters, also lacks cross-reactivity with antibodies directed against human BP180 [59]. To assess the pathogenicity of human anti-BP180 autoantibodies, we generated a novel mouse strain in which the murine BP180NC14A was replaced with the homologous human BP180NC16A epitope cluster region [38]. The humanized NC16A (NC16+/+) mice injected with anti-BP180NC16A autoantibodies develop BP, and the disease development depends on complement, mast cells and neutrophils (Fig. 1).
Fig. 1Proposed mechanism of subepidermal blister formation in humanized NC16A mouse model of BP. Subepidermal blistering is an inflammatory process involved in following steps: a anti-BP180 autoantibodies are injected into NC16A mice; b pathogenic antibodies bind to the pathogenic epitope of BP180 antigen in basal keratinocytes (BK); c the molecular interaction between BP180 antigen and anti-BP180 IgG activates the complement system (C′) as evidenced by BMZ deposition of mouse C3; d C′ activation products C3a and C5a cause mast cells (MC) to degranulate; e MC activation leads to neutrophil (PMN) recruitment; f infiltrating PMNs bind to the BP180-anti-BP180 immune complex via the molecular interaction between Fcγ receptors on neutrophils and the Fc domain of anti-BP180 IgG. Activated PMNs release proteolytic enzymes; g proteolytic enzymes degrade BP180 and other extracellular matrix proteins, leading to dermal–epidermal junction separation; h pathogenic antibody-injected mice develop clinical blisters
Using the NC16A+/+ mice and BP patients’ sera, we mapped the pathogenic epitope of BP180 in vivo [39]. IgG passive transfer experiments identified one pathogenic epitope (referred to as BP180NC16A2.5). BP180NC16A2.5-specific autoantibodies induced subepidermal blisters in the NC16A+/+ mice, and these blisters were blocked by pretreatment with recombinant BP180NC16A2.5. We also found that the NC16A2.5 pretreated mice had significantly reduced levels of BMZ-bound and circulating pathogenic antibodies and showed reduced complement activation, mast cell degranulation and neutrophil infiltration. These results suggest that targeting the pathogenic epitope specifically could be a new therapeutic strategy to treat BP.
A second humanized animal model has been developed by Nishie et al. [42, 43] that replaces the mouse BP180 (COL17) with the human analogue. Upon injection of human anti-BP180 autoantibodies, these mice reproduce the DEJ separation at the lamina lucida, the deposition of human IgG along the DEJ, and an inflammatory cell infiltrate consisting of neutrophils and eosinophils seen in human disease. The pathogenic effects of the autoantibody injection are ablated by pretreatment with a 77-amino acid peptide fragment of COL17 NC16A, referred to as R1. These studies further support the therapeutic potential of employing decoy peptides to block the pathogenic epitope.
Relevance of murine and humanized passive transfer models to human BP
Both in vitro and in vivo data demonstrate that BP180 is the target for pathogenic autoantibodies in BP (Table 1). While clinical human BP and experimental murine and humanized murine BP closely mimic each other at the clinical, histological, and immunological levels, the IgG passive transfer models do not reflect the large number of eosinophils typically found in the inflammatory infiltrate of human BP lesional skin. Some patients do exhibit neutrophil-rich pemphigoid, indicating that the neutrophil-mediated blistering observed in these mouse models may be one of several disease mechanisms that contribute to the formation and persistence of subepidermal blisters. The presence of a neutrophil-rich inflammatory infiltrate in the two humanized mouse models, as well as in the aforementioned hamster model, lend credence to a neutrophil-mediated disease mechanism. Furthermore, findings that in vitro DEJ separation induced by human BP autoantibodies specific for BP180NC16A depends on neutrophils [51] and neutrophil-derived elastase and gelatinase B [50], and that BP180 degradation by human BP blister fluid depends on neutrophil elastase activity [57] support this theory. Taken together, these findings strongly suggest that like the IgG passive transfer model of BP, neutrophils may be responsible for subepidermal blister formation in human BP, at least in those patients who show neutrophil infiltration in their lesional/perilesional skin. The humanized mouse models do not yet offer insight to an eosinophil-mediated mechanism of disease progression, leaving the role of these inflammatory cells, if there is any, yet to be discovered. It is possible that these established mouse models, without additional experimental manipulations, may not be able to fully duplicate human BP pathology related to eosinophil infiltration; therefore, they may not be appropriate to study the role of eosinophils in BP.
Table 1In vitro and in vivo evidence of pathogenicity of anti-BP180 antibodiesSystemAntibodies usedReferenceIn vitro Human skin sectionBP sera[13]Anti-BP180NC16A autoantibodies[51]Rabbit anti-BP180NC16A IgGIn vivo Wild-type miceRabbit anti-murine BP180 IgG[30] HamsterRabbit anti-hamster BP180 IgG[60] Humanized BP180 miceAnti-BP180NC16A autoantibodies[38, 39] Humanized NC16A miceAnti-BP180NC16A autoantibodies[42, 43]
Zone et al. [64] reported that IgE hybridoma to LABD97 antigen, a component of the shed ectodomain of BP180, when injected subcutaneously in SCID mice with engrafted human skin, induced eosinophil infiltration and histological blisters in engrafted human skin. Fairley et al. [12] also reported that total IgE isolated from BP sera, when injected into human skin grafted onto athymic nude mice, triggered a dermal infiltrate composed of neutrophils, eosinophils, and histological separation of the epidermis from the dermis. These findings implicate eosinophils in BP; however, a direct link between eosinophil recruitment and blistering in these human skin graft models needs to be established.
Conclusions
The murine IgG passive transfer model of BP developed by Liu et al. [30] has provided an invaluable tool for the dissection of the mechanism of disease progression. The development of humanized murine models of BP now provides unprecedented insight to the pathogenic role of the autoantibodies that mediate disease. Initial observations with these humanized models indicate that humanized mouse BP exhibits phenotypic and immunological similarities with murine and human BP. Additionally, these humanized models enabled the first identification of pathogenic epitopes in human disease. Blocking of the pathogenic epitope with decoy peptides, if they do not enhance the autoimmune responses, presents a novel clinical therapeutic strategy that may be very effective in abolishing the pathogenic effects of disease.

hemidesmosome
autoimmune disease
basement membrane
humanized animal model
inflammation