Vitamin D deficiency is associated with tuberculosis disease in British children.

BACKGROUND: Basic science, epidemiological and interventional research supports a link between vitamin D and tuberculosis (TB) immunity, infection and disease. We evaluated the association between vitamin D levels and TB infection and disease in UK children recruited to the National Institute for Health Research IGRA Kids Study (NIKS).METHODS: Children presenting between 2011 and 2014 were eligible if they had history of exposure to an adult case with sputum smear/culture-positive TB, or were referred and diagnosed with TB disease. Children were assessed at baseline and at 6-8 weeks for immunological evidence of TB infection (interferon-gamma release assay and/or tuberculin skin test) and evidence of TB disease. Some centres routinely measured total 25-hydroxy vitamin D (25-OHD) levels.RESULTS: A total of 166 children were included. The median 25-OHD levels were higher in non-infected children (45.5 nmol/l) than in those with tuberculous infection (36.2 nmol/l) and TB disease (20.0 nmol/l). The difference between TB infection and disease was statistically significant (P < 0.001). By logistic regression, lower vitamin D levels were associated with TB disease among participants with infection or disease, with no evidence of confounding by age, sex, bacille Calmette-Guérin vaccination status, ethnicity, non-contact referral, season or centre.CONCLUSION: Children with TB disease had lower vitamin D levels than children with infection. Implications for prevention and treatment remain to be established.

Equitable tuberculosis care in the North West of England: analysis of tuberculosis cohort review data.

BACKGROUND: In the United Kingdom, tuberculosis (TB) predominantly affects the most deprived populations, yet the extent to which deprivation affects TB care outcomes is unknown. METHODS: Since 2011, the North West TB Cohort Audit collaboration has undertaken quarterly reviews of outcomes against consensus-defined care standard indicators for all individuals notified with TB. We investigated associations between adverse TB care outcomes and Index of Multiple Deprivation (IMD) 2010 scores measured at lower super output area of residence using logistic regression models. RESULTS: Of 1831 individuals notified with TB between 2011 and 2014, 62% (1131/1831) came from the most deprived national quintile areas. In single variable analysis, greater deprivation was significantly associated with increased likelihood of the completion of a standardised risk assessment (OR 2.99, 95%CI 5.27-19.65) and offer of a human immunodeficiency virus test (OR 1.72, 95%CI 1.10-2.62). In multivariable analysis, there were no significant associations. CONCLUSIONS: TB patients in the most deprived areas had similar care indicators across a range of standards to those of individuals living in the more affluent areas, suggesting that the delivery of TB care in the North West of England is equitable. The extent to which the cohort review process contributes to, and sustains, this standard of care deserves further study.

Surgical treatment of metastases to the pancreas.

BACKGROUND AND AIMS: Metastases to the pancreas are rare and their surgical treatment is not well reported. We present a considerable experience from a single centre analysing various prognostic factors. METHODS: Data were collected on 13 cases who underwent surgery between 1988 and 2002. Since 1997, data have been recorded prospectively on a dedicated database. Clinical and histopathological factors were reviewed. RESULTS: There were two women and 11 men with a median age of 62 years (range 40-73). There were seven cases of renal cell carcinomas, three colorectal carcinomas, two sarcomas and one lung carcinoma. A prolonged disease-free interval from primary surgery was characteristic for renal cell carcinoma cases (median = 10.8 years). The operative procedures performed included seven pancreatoduodenectomies, four total and two distal pancreatectomies. The operative mortality and morbidity was 7.7% and 46.1% respectively. The overall one- and two-year survival was 78.8% and 54% respectively. Median survival for renal cell carcinoma was 30.5 months and for non-renal cell carcinoma was 26.4 months (p = 0.76). CONCLUSIONS: Pancreatectomy should be considered for metastases to the pancreas in the absence of generalised metastatic disease. However, decision making and experience should be concentrated in centres with significant familiarity of this approach.

Treatment of hydatid disease of the liver. Evaluation of a UK experience.

BACKGROUND: Hydatid disease of the liver though endemic in many countries, is rare in the UK. We evaluated a 16-year experience of treating hydatidosis using a management protocol combining surgery with anti-scolicidals. PATIENTS AND METHODS: There were 30 patients. 14 (47%) males, median age 41 (range 25-72) years, of whom 21 (70%) were symptomatic. Diagnosis was by serological tests and imaging. All had disease confined to the liver and received peri-operative anti-scolicidal drug therapy. RESULTS: The initial 4 (13%) patients received praziquantel combined with albendazole for 2 weeks and the following 26 (87%) patients received two cycles of albendazole 400 mg twice daily for 28 days, with a 14-day break in between. However, 2 (7%) patients could not tolerate albendazole, one due to GI side effects and the other developed deranged liver functions. These 2 patients subsequently received praziquantel for 2 weeks. All patients underwent surgery. Subtotal cystectomy was carried out on 29 (96%) patients and 1 patient required a segmentectomy. Cystobiliary communications were identified in 15 (50%) of patients which were oversewn using fine absorbable sutures. Of these, 7 had the bile ducts decompressed using a T tube, with only 1 developing a post-operative bile leak. In comparison, 8 were not drained of which 6 leaked (p = 0.03). The median post-operative hospital stay was 8 days (range 5-24). Patients who developed post-operative bile leaks, however, needed prolonged abdominal drainage for a median of 21 days (range 18-24). Two (7%) patients developed histologically proven recurrent disease. The median follow-up was 56 months (range 3-87). CONCLUSION: Surgery combined with anti-scolicidal therapy proved effective. Cystobiliary communications are common and, when identified, should result in the biliary system being drained, to avoid post-operative bile leaks.

Analysis of marginal donor parameters in liver transplantation for primary biliary cirrhosis.

The shortage in cadaveric donor livers is pushing the transplant centers to expand the pool by using "marginal" donors. Primary biliary cirrhosis (PBC) remains an important indication for transplantation. We conducted a retrospective analysis of prospectively collected data in a well-defined group of patients with PBC where 301 consecutive donor-PBC recipient pairs transplanted were analyzed to identify donor and operative factors influencing recipient outcome. Mean follow-up was 56 months. The 1-, 3- and 5-year actuarial patient and graft survival was 93.97%, 90.64%, and 81.75%, and 85.49%, 82.57%, and 75.21%, respectively. Factors showing influence in decreased total patient survival were recipient old age (P = 0.003) and low recipient albumin (P = 0.01). However, the only variables showing an association with decreased patient survival within 90 days are old donor age (P = 0.002) and high donor body weight (P = 0.03) or high body mass index (BMI) (P = 0.055). Cold ischaemic time (CIT) of 18 hours showed statistical significance in patient survival (P = 0.025). Obesity did have a significant adverse impact on survival compared with normal or overweight donors (BMI < 30), decreasing survival by 50% at 5 years. In conclusion, this study of several factors considered "marginal" for transplantation in a recipient population with predictable liver disease (PBC), donor BMI and age were shown to be associated with decreased graft and patient survival.

Liver resection for colorectal metastases.

BACKGROUND: Colorectal cancer is the second commonest malignancy in the UK. Metastases to the liver occur in greater than 50% of patients and remain the biggest determinant of outcome in these patients. Liver resection is a safe procedure that achieves good long-term survival, but surgery has traditionally been limited to select groups of patients. The improved outcome suggests that more patients could benefit from resection if more was known of what criteria are predictive of a good outcome. PATIENTS AND METHODS: A retrospective analysis was performed on all patients undergoing surgical resection of the liver for colorectal metastases between March 1989 and March 2001 in the Birmingham Liver Unit. RESULTS: During this period, 212 liver resections for colorectal cancer metastases were performed in 82 females and 130 males. The median follow-up was 16 months with an overall actuarial survival of 86% at 1 year, 54% at 3 years, and 28% at 5 years. The peri-operative mortality was 2.8%. The number and timing (metachronous or synchronous) of metastatic lesions, the gender of the patient, pathological staging of the primary lesion or surgical resection margins had no significant influence on survival. Patients with lesions less than 5 cm in size had a significantly prolonged survival compared with patients with lesions greater than 5 cm in size (P < 0.004). CONCLUSIONS: Liver resection is the only curative treatment for patients with colorectal metastases. The long-term survival reported in patients with resected colorectal metastases confined to the liver is comparable to primary surgery for solid gastrointestinal tumours. Every attempt must be made to increase the availability of liver resection to patients with hepatic metastases from colorectal cancer.

A 10-year experience of complex liver trauma.

BACKGROUND: Liver trauma is a relatively rare surgical emergency but mortality and morbidity rates remain significant. It is likely that surgeons outside specialist centres will have limited experience in its management; therefore best practice should be identified and a specialist approach developed. METHODS: Data collected from 52 consecutive patients over a 10-year interval were examined to identify best practice in the management of these injuries. RESULTS: The majority of injuries occurred as a result of road traffic accidents; 39 (75 per cent) of the 52 patients were stable at presentation to the referring hospital. In 36 patients (69 per cent) the liver injury was a component of multiple trauma. Ultrasonography, computed tomography or no radiological investigation was used in the referring hospital in 18 (35 per cent), 25 (48 per cent) and nine (17 per cent) patients respectively. Operative management was undertaken in the referring hospital in 26 patients (50 per cent). The overall mortality rate was 23 per cent (12 of 52 patients), and increased with increasing grade of severity. Eight of 26 patients managed surgically at the referring hospital died, compared with four of the 26 patients managed without operation (P not significant). The median time from arrival at the referring hospital to operation was 4 h for haemodynamically stable patients and 3 h for those who were haemodynamically unstable. CONCLUSION: Most patients with liver trauma can be managed conservatively. Operative management carried out in non-specialized units is associated with high mortality and morbidity rates. Abdominal injuries should raise a high index of suspicion of liver injury, and the data suggest that computed tomography of the abdomen should precede laparotomy (even in some haemodynamically unstable patients) to facilitate discussion with a specialist unit at the earliest opportunity.

The emergence of resistant pneumococcal meningitis--implications for empiric therapy.

BACKGROUND: Following the emergence of penicillin and cephalosporin resistant pneumococcal meningitis in the United States, inclusion of vancomycin in empiric therapy for all suspected bacterial meningitis was recommended by the American Academy of Pediatrics. Few data are available to evaluate this policy. AIMS: To examine the management and clinical course in relation to antibiotic therapy of a large unselected cohort of children with pneumococcal meningitis in a geographic area where antibiotic resistance has recently increased. METHODS: Retrospective review of all cases of pneumococcal meningitis in a defined population (Sydney, Australia), 1994-99. RESULTS: A total of 104 cases without predisposing illnesses were identified; timing of lumbar puncture (LP) was known in 103. Resistance to penicillin increased from 0 to 20% over the study period. Only 57 (55%) had an early LP (prior to parenteral antibiotics); 55 (96%) had organisms on Gram stain. Severe disease (intensive care admission or death) increased significantly from 57 cases with early LP (28%) to 33 with delayed LP (42%) to 13 with no LP (62%). Evidence of pneumococcal infection was available within 24 hours in 85% of those with delayed or no LP. Outcome was not related to empiric vancomycin use, which increased from 5% prior to 1998 to 48% in 1999. CONCLUSION: LP is frequently delayed in pneumococcal meningitis. Based on disease severity, empiric vancomycin is most justified when LP is deferred. If an early LP is done, vancomycin can be withheld if Gram positive diplococci are not seen.

The use of liver grafts from donors with bacterial meningitis.

BACKGROUND: The shortage of suitable donors for transplantation is a worldwide problem. The use of cadaveric donors with bacterial meningitis may be associated with an increased risk of sepsis. We report the results of orthotopic liver transplantation (OLT) from 33 such donors between 1989 and 1999. METHODS: The hospital records of recipients from cadaveric donors with meningitis (study group) were retrospectively reviewed and compared with matched recipients from cadaveric donors dying from causes other than meningitis (recipient-matched control group). RESULTS: A total of 34 recipients underwent 21 whole, 10 reduced, and 3 split liver transplants from 33 cadaveric donor livers with bacterial meningitis. The donor meningitis pathogens were Neisseria meningitidis (n=14), Streptococcus pneumoniae (n=4), Haemophilus influenzae (n=1), Streptococcus species (n=2), and unknown (n=12). Twenty-seven patients had an elective OLT and seven patients had an emergency OLT. Adequate antimicrobial therapy before organ procurement and after transplant was administrated. The mean posttransplant follow-up was 37 months (range: 1 day-106 months). There was no difference in recipient and graft survival rates between the study and the recipient-matched groups. In the study group, there were no infectious complications caused by the meningeal pathogens. Overall patient survival rates were 79%, 76%, 72%, and 72% at 1, 6, 12, and 60 months, respectively. Graft survival was 77%, 70%, 65%, and 65% at 1, 6, 12, and 60 months, respectively. The survival rate in elective cases was significantly better than emergency cases (P<0.05). CONCLUSION: Liver transplantation from donors with bacterial meningitis is a safe procedure provided both donors and recipients receive adequate antimicrobial therapy.

Type of donor aortic preservation solution and not cold ischemia time is a major determinant of biliary strictures after liver transplantation.

The development of biliary strictures (BSs) after liver transplantation (LT) continues to affect 10% to 30% of patients, causing substantial morbidity. The cause of BSs is multifactorial, including technical, immune, and, in particular, ischemic factors. The importance of adequate flushing of the peribiliary arterial tree has been stressed. We hypothesized that high-viscosity (HV) preservation solutions in the donor do not completely flush the small donor peribiliary plexus, leading to inadequate preservation of the bile ducts and posttransplant BSs. To test this hypothesis, we retrospectively compared the incidence of BSs in 2 groups of adults undergoing LT using different types of aortic preservation solution in the donor: group 1 (n = 24), low-viscosity (LV) Marshall solution; and group 2 (n = 27), HV University of Wisconsin (UW) solution. All donors in both groups received additional portal flushes with UW. All LTs were performed between November 1995 and August 1998 at 2 centers by the same surgeon, eliminating a technical bias. Terminal duct-to-duct anastomosis was performed in all recipients except 1 patient in group 1, who underwent a bile duct-to-jejunum anastomosis. BSs were first suspected on clinical and biochemical grounds and then confirmed by endoscopic retrograde cholangiopancreatography. Identical medical protocols were used in all patients. One-year patient survival rates in groups 1 and 2 were 92% and 100%, respectively (P =.9). One-year graft survival was identical to patient survival. The incidence of BSs in group 1 was 4.1% (1 of 24 patients), compared to 29.7% in group 2 (8 of 27 patients; P =.02). The BS in group 1 occurred 4 months post-LT and was anastomotic. BSs in group 2 occurred between 1 and 12 months post-LT and were anastomotic, extrahepatic, intrahepatic, or combined intrahepatic and extrahepatic. There were no significant differences in the following factors between groups 1 and 2: donor age, local versus imported liver, split-liver or full-liver transplantation, incidence of multiple vessels in the donor liver, indications for LT, recipient age, T-tube versus no T-tube, post-LT peak aspartate aminotransferase level, and treatment for rejection. There was no hepatic artery thrombosis or primary nonfunction in either group. Interestingly, cold ischemia time (CIT) was longer in group 1, which had the least incidence of BSs (692 +/- 190 v 535 +/- 129 minutes in group 2; P =.001). Follow-up was longer in group 1 (28.9 +/- 8.3 v 15.6 +/- 8 months in group 2; P =.0001). Preservation costs per procurement were 1.9 times greater in the UW group than in the Marshall group. Donor aortic flushing with an HV preservation solution leads to more frequent BSs compared with an LV preservation solution. The impact of preservation solution outweighs the previously described deleterious impact of prolonged CIT. Mixed preservation solution (Marshall solution in the aorta, UW solution in the portal vein) might protect against BS formation while providing optimal liver graft preservation, function, and survival despite a mean CIT longer than 10 hours.

Delayed hepatic artery thrombosis in adult orthotopic liver transplantation-a 12-year experience.

BACKGROUND: Although the clinical features of early hepatic artery thrombosis (HAT) are well defined, the features of delayed (more than 4 weeks after transplantation) hepatic artery thrombosis are less clearly defined. The aim of our study was to identify risk factors, clinical presentation, and outcome of management of delayed hepatic artery thrombosis (HAT) after liver transplant (LTx). METHODS: An analysis of prospectively collected data of all patients transplanted from 1986 to 1998 was performed. The importance of recipient (age, sex, primary indication for LTx, cytomegalovirus status, and intraabdominal sepsis) and donor factors (donor age, cold ischemia time, and donor cytomegalovirus status), modes of presentation, and outcome of treatment (biliary reconstruction/stenting, regraft, vascular reconstruction, observation) were analyzed. RESULTS: Delayed HAT was seen in 31/1097 adult LTx recipients (incidence 2.8%). No recipient or donor factors were identified as risk factors. A total of 16 patients were symptomatic at presentation (HAT diagnosed on abdominal ultrasound). Six patients had recurrent episodes of cholangitis, four had cholangitis with a stricture, four had cholangitis and intrahepatic abscesses, and two had bile leaks. Biliary reconstruction was done in six patients (all of whom subsequently required a regraft), vascular reconstruction was performed in two patients (one regrafted and one died shortly after), four patients with cholangitis and stricture on presentation had a biliary stent (all four were later regrafted). A total of 16 patients were regrafted, 9 are alive, 5 died within 6 months (septic at time of LTx), 1 died after 1 year, and 1 died after 2 years. Fifteen patients were asymptomatic and detected on routine screening. 5 have remained asymptomatic and are still alive, 1 developed a biliary stricture that was stented and is alive 105 months later, 4 had recurrence of the original disease, 3 developed progressive graft failure and were listed for transplant but died before regraft due to overwhelming sepsis and hepatic encephalopathy. Two patients died due to nonbiliary sepsis. CONCLUSIONS: Delayed HAT is a rare complication of LTx that may present with biliary sepsis, or remain asymptomatic. Biliary or vascular reconstructions do not increase graft survival. Of the patients who were clinically silent on presentation, 20% developed progressive graft failure requiring a second transplant. A total of 33% survived in the long-term without a second transplant. Ongoing severe sepsis at the time of regraft results in poor survival.

Ten-year experience with paediatric lymph node tuberculosis in Port Moresby.

This is a descriptive study of short-course chemotherapy in children with nodal tuberculosis at Port Moresby General Hospital (PMGH). Between 1 August 1989 and 31 December 1997 5248 children were started on TB treatment. In the retrospective study 427 children were treated for lymph node TB up to 31 December 1996. Of these, 207 definitely completed the treatment and 24 (11.6 per cent) of them were known to have relapsed up to the end of 1997. In the prospective study 179 children with lymph node TB were enrolled between 1 January 1997 and 31 December 1997. Of these, 97 definitely completed the treatment and 10 (10.6 per cent) were known to have relapsed during a follow-up period of between 1 and 2 years.

A randomized double-blind comparative study of mycophenolate mofetil and azathioprine in combination with cyclosporine and corticosteroids in primary liver transplant recipients.

Acute hepatic allograft rejection occurs in approximately 50% to 60% of the patients undergoing liver transplantation. In this study, we compared the rate of acute rejection in liver transplant recipients randomized in a double-blind comparative study to treatment with mycophenolate mofetil (MMF) or azathioprine (AZA), both in combination with cyclosporine and corticosteroids. Five hundred sixty-five primary liver transplant recipients were randomly assigned to treatment with MMF, 1 g twice daily intravenously followed by 1.5 g twice daily orally (n = 278), or AZA, 1.0 to 2.0 mg/kg/d intravenously followed by oral administration (n = 287), in combination with cyclosporine and corticosteroids. Patients were followed up for at least 1 year, and efficacy analysis was based on intent-to-treat methods. Acute rejection was defined according to the Banff histological criteria. The two study groups were balanced for demographic and clinical baseline characteristics. The incidence of acute rejection or graft loss was 47.7% in the AZA patients and 38.5% in the MMF patients (P <.03). The incidence of biopsy-proven and treated rejection censoring for graft loss was 40.0% in the AZA group versus 31.0% in the MMF group (P <.06). Steroid-resistant rejection requiring treatment with either OKT3 or antithymocyte globulin occurred in 8.2% of AZA patients versus 3.8% in MMF patients (P <.02). Patient and graft survival rates at 1 year posttransplantation were 85.4% in the AZA group and 85.3% in the MMF group (P = not significant). MMF was superior to AZA in preventing acute rejection in the first 6 months posttransplantation. MMF and AZA were equivalent in preventing graft loss at 1 year, and the safety profiles between the two immunosuppressive agents were similar.

Hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the sixth most common cancer of men and eleventh most common cancer of women world-wide. However, because almost every individual who develops liver cancer dies of the disease, HCC is the third most common cause of the cancer deaths in men and seventh most common in women. The treatment of choice for hepatocellular carcinoma remains surgical resection or liver transplantation, in carefully selected cases. In patients with hepatocellular carcinoma not amenable to surgical intervention a variety of different therapeutic interventions have been investigated. These include direct ablation of the tumour using agents such as ethanol or acetic acid, transcatheter arterial chemoembolization, or systemic chemotherapy. The evaluation of their efficacy is compromised by the paucity of adequately powered randomised clinical trials. The main challenge facing the research community over the next decade is to prioritise the most promising treatments and take these forward into multicentre controlled trials. Even if these fail to improve results, they will help reduce the variation in clinical practice by eliminating anecdotal treatment.

Liver transplantation for polycystic liver disease.

Polycystic liver disease (PLD) may provoke massive hepatomegaly and severe physical and social handicaps. Data on orthotopic liver transplantation (OLT) for PLD are rare and conflicting. Conservative surgery (resection or fenestration) is indicated for large single cysts, but its value for small diffuse cysts is questionable. In addition, conservative surgery is not devoid of morbidity and mortality. OLT offers the prospect of a fully curative treatment, but controversy remains because those patients usually have preserved liver function. Thus, we reviewed our experience with OLT for PLD. Sixteen adult women underwent OLT for small diffuse PLD between 1990 and 1999. Mean age was 45 years (range, 34 to 56 years). Fourteen patients had combined liver and kidney cystic disease, but only 1 patient required combined liver and kidney transplantation, whereas 13 patients underwent OLT alone. Two patients had isolated PLD. Indications for transplantation were massive hepatomegaly causing physical handicaps (n = 16), social handicaps (n = 16), malnutrition (n = 4), and cholestasis and/or portal hypertension (n = 5). OLT caused no technical difficulty in 15 of 16 patients (surgery duration, 6.8 hours; range, 5 to 8 hours), with blood transfusions of 7.9 units (range, 0 to 22 units). One patient who underwent attempted liver-mass reduction pre-OLT died of bleeding and pulmonary emboli. Native liver weight was 10 to 20 kg. Posttransplantation immunosuppression consisted of cyclosporine or FK506, azathioprine, and steroids (discontinued at 3 months). Morbidity included biliary stricture (2 patients), revision for bleeding and hepatitis (1 patient), pneumothorax and subphrenic collection (1 patient), and tracheostomy (1 patient). One patient died of lung cancer 6 years posttransplantation. Both patient and graft survival rates are 87.5% (follow-up, 3 months to 9 years). Of 15 patients who underwent OLT alone, only 1 patient needed a kidney transplant 4 years after OLT. Kidney function has remained satisfactory in the other patients despite the use of cyclosporine or FK506 (last follow-up creatinine level, 1.55 mg/dL; range, 0.80 to 2.85 mg/dL). OLT had a dramatic impact on daily quality of life, enabling these patients to go back to a fully active life style. OLT offers the chance of a definitive treatment in patients with extensive, small, diffuse PLD that has evolved into severely handicapping hepatomegaly. In contrast to previous studies, combined liver and kidney transplantation is rarely needed. Patient symptoms and chances of definitive palliation offered by OLT must be balanced against the risks of transplantation and lifelong commitment to immunosuppression.

Morphogenesis of primary human biliary epithelial cells: induction in high-density culture or by coculture with autologous human hepatocytes.

Although the control of biliary ductular morphogenesis has received some attention particularly using isolated rat biliary epithelial cell models, the regulation of human bile duct formation is not well defined. In the present study, using a 3-dimensional culture model comprising primary human biliary epithelial cells (BECs) and coculture with primary human hepatocytes, we have sought to define the factors involved. We have shown that primary human BECs can be expanded on collagen gels in the absence of growth factors or serum. When plated in high density in double collagen gels, BECs established 3-dimensional structures that subsequently developed into well differentiated polarized luminal ducts. This morphogenic response occurred in the absence of hepatocyte growth factor (HGF) and epidermal growth factor. Strikingly, the addition of growth factors (in the presence of serum) resulted in loss of polarity although the cells retained growth responses to both factors. Coculture of BECs with autologous human hepatocytes enhanced the ability of low-density BECs to undergo ductulogenesis. This effect was mimicked by addition of conditioned medium from previous hepatocyte-BEC cocultures. These findings indicate that for human biliary ductular morphogenesis, epithelial cell-cell interactions are required but that mesenchymally derived factors such as HGF may not be important.