HCV NS3 sequencing as a reliable and clinically useful tool for the assessment of genotype and resistance mutations for clinical samples with different HCV-RNA levels.

OBJECTIVES: This study aims to evaluate the reliability and clinical utility of NS3 sequencing in hepatitis C virus (HCV) 1-infected patients who were candidates to start a PI-containing regimen. METHODS: NS3 protease sequencing was performed by in-house-developed HCV-1 subtype-specific protocols. Phylogenetic analysis was used to test sequencing reliability and concordance with previous genotype/subtype assignment by commercial genotyping assays. RESULTS: Five hundred and sixty-seven HCV plasma samples with quantifiable HCV-RNA from 326 HCV-infected patients were collected between 2011 and 2014. Overall, the success rate of NS3 sequencing was 88.9%. The success rate between the two subtype protocols (HCV-1a/HCV-1b) was similarly high for samples with HCV-RNA >3 log IU/mL (>92% success rate), while it was slightly lower for HCV-1a samples with HCV-RNA ≤3 log IU/mL compared with HCV-1b samples. Phylogenetic analysis confirmed the genotype/subtype given by commercial genotyping assays in 92.9% (303/326) of cases analysed. In the remaining 23 cases (7.1%), 1 was HCV-1g (previously defined as subtype 1a), 1 was HCV-4d (previously defined as genotype 1b) and 1 was HCV-1b (previously defined as genotype 2a/2c). In the other cases, NS3 sequencing precisely resolved the either previous undetermined/discordant subtype 1 or double genotype/subtype assignment by commercial genotyping assays. Resistance-associated variants (RAVs) to PI were detected in 31.0% of samples. This prevalence changed according to PI experience (17.1% in PI-naive patients versus 79.2% in boceprevir/telaprevir/simeprevir-failing patients). Among 96 patients with available virological outcome following boceprevir/telaprevir treatment, a trend of association between baseline NS3 RAVs and virological failure was observed (particularly for HCV-1a-infected patients: 3/21 failing patients versus 0/22 achieving sustained virological response; P = 0.11). CONCLUSIONS: HCV-NS3 sequencing provides reliable results and at the same time gives two clinically relevant pieces of information: a correct subtype/genotype assignment and the detection of variants that may interfere with the efficacy of PI.

Long-term maintenance of sustained virological response in liver transplant recipients treated for recurrent hepatitis C.

BACKGROUND: The recurrence of hepatitis C viral infection is common after liver transplant, and achieving a sustained virological response to antiviral treatment is desirable for reducing the risk of graft loss and improving patients' survival. AIM: To investigate the long-term maintenance of sustained virological response in liver transplant recipients with hepatitis C recurrence. METHODS: 436 Liver transplant recipients (74.1% genotype 1) who underwent combined antiviral therapy for hepatitis C recurrence were retrospectively evaluated. RESULTS: The overall sustained virological response rate was 40% (173/436 patients), and the mean follow-up after liver transplantation was 11±3.5 years (range, 5-24). Patients with a sustained virological response demonstrated a 5-year survival rate of 97% and a 10-year survival rate of 93%; all but 6 (3%) patients remained hepatitis C virus RNA-negative during follow-up. Genotype non-1 (p=0.007), treatment duration >80% of the scheduled period (p=0.027), and early virological response (p=0.002), were associated with the maintenance of sustained virological response as indicated by univariate analysis. Early virological response was the only independent predictor of sustained virological response maintenance (p=0.008). CONCLUSIONS: Sustained virological response achieved after combined antiviral treatment is maintained in liver transplant patients with recurrent hepatitis C and is associated with an excellent 5-year survival.

Hydroxyurea-induced interstitial pneumonitis: case report and review of the literature.

Hydroxyurea is a cytotoxic agent widely used in the treatment of myeloproliferative disorders. It is considered a-well-tolerated antineoplastic drug, with a dose-related bone marrow suppression as main adverse effect. This report describes a patient with essential thrombocythemia who developed an interstitial pneumonitis and respiratory failure within 4 years from beginning therapy with hydroxyurea (HU). After discontinuing of HU. both clinical and radiological resolution of pneumonitis occurred. In conclusion, HU-induced pulmonary toxicity is a potentially life-threatening side effect.

Conversion from twice-daily to once-daily Tacrolimus administration in liver transplant patient: results of long term follow-up.

OBJECTIVES: The aim of this study was to assess the long term effects of once-daily tacrolimus (OD-TAC) in a cohort of stable liver recipients converted from the twice daily tacrolimus (TD TAC), with a particular attention on the possible effects on renal function. PATIENTS AND METHODS: Between September 2008 and September 2010 conversion from TD-TAC to OD-TAC was proposed in adult stable liver transplant recipients who were followed as outpatients in our Transplant centre. Conversion from TC-TAC to OD-TAC was based on a 1 mg: 1 mg proportion. Tacrolimus through levels, laboratory parameters, metabolic disorders and any adverse events were evaluated at 1, 3, 6, 12 and 24 months after conversion. Renal function was evaluated using creatinine plasma levels and estimated glomerular filtration rate (GFR) derived from the Modification of Diet in Renal Disease (MDRD). Analysis of variance and t test for paired data were utilised for the comparison of the results obtained at the scheduled controls. RESULTS: Sixty-five patients were enrolled in the study (50 males, 15 females, mean age 59±8 years). Median time since liver transplant (LT) was 39 months (range: 6 to 83 months). All patients were followed for a minimum of 12 months. Ninety per cent of patients stabilized their blood levels within 45 days. Liver function, glucose and plasma lipids concentration and arterial blood pressure remained stable during the study. Renal function improved during the 24 months of follow-up. No adverse events or acute rejection episodes were recorded during the study. CONCLUSIONS: Considering the advantage on patient compliance, the equivalent efficacy and the adequate safety of OD-TAC formulation may represent a useful option in liver transplant patients, with a possible advantage on renal function.

Occurrence of chronic renal failure in liver transplantation: monitoring of pre- and posttransplantation renal function.

The aim of our study was to evaluate the occurrence of middle and long-term chronic renal failure (CRF) after orthotopic liver transplantation (OLT) in relation to acute renal failure (ARF). We prospectively monitored 75 patients, studying renal function on the basis of serum creatinine and glomerular filtration rate as estimated using the Modification of Diet in Renal Disease formula before as well as 1,6, and 12 months after OLT. The prevalence of ARF was 56% classified by the Acute Kidney injury Network criteria (52% stage 1, 29% stage 2, and 19% stage 3). The occurrences of CRF were 18.6% (11/59), 11.5% (6/52), and 14% (6/43) at 1, 6, and 12 months after OLT, respectively. The occurrence of CRF before OLT was 14.7%. We did not find any association between ARF and post-OLT CRF. The most relevant result of our study was the association between CRF at 6 and 12 months after transplantation with pre-OLT CRF on univariate and multivariate analysis. We suggest that evaluation of pre-OLT renal function should always be considered in the follow-up of liver transplant patients. Pre-OLT renal dysfunction must be recognized to be a risk factor for post-OLT CRF, representing important criterion to define specific therapeutic interventions to reduce patient morbidity and mortality.

Metabolic syndrome and cardiovascular risk after liver transplantation: a single-center experience.

Excessive weight gain, hypertension, hyperlipidemia, and diabetes are frequently observed among orthotopic liver transplantation (OLT) patients. These alterations, which are probably multifactorial in origin, contribute to posttransplantation metabolic syndrome (PTMS), which increases the risk of cardiovascular events. We assessed the prevalence of PTMS (diagnosed according to modified NCEP Adult Treatment Panel III criteria) in 156 OLT patients undergoing regular follow-up after transplantation (median 68 months; range, 6 to 234 months). Several pre- and post-OLT data were collected to identify the factors associated with the presence of PTMS which was found in 28% of cases. The only independent predictive factors for PTMS were diabetes mellitus and patients who were overweight or obese before-OLT. The prevalence of PTSM was lower among patients on tacrolimus immunosuppression. In our population, 21% of patients showed a high cardiovascular risk score with a 4% incidence of cardiovascular events, which was higher among subjects with PTMS. Close follow-up is mandatory to prevent the development of PTMS mainly among overweight and diabetic patients before transplantation.

Acute renal failure in liver transplant recipients: role of pretransplantation renal function and 1-year follow-up.

Chronic renal failure and acute renal failure (CRF and ARF) are common complications after orthotopic liver transplantation (OLT) that adversely affect patient survival. Many factors influence the development of ARF in the OLT setting. In a previous study we reported an association between ARF and the development of CRF at 1 month after OLT. The aims of our study were to evaluate the influence of ARF on short-, middle-, and long-term renal function after OLT and its influence on 1-year survival of patients and grafts. Fourty-four patients who underwent deceased donor OLT between August 2008 and August 2010 were evaluated pretransplantation, in the perioperative period, and at 1, 6, and 12 months posttransplantation. ARF was associated with CRF at 1 month post-OLT, whereas no association was observed at 6 and 12 months post-OLT. The development of CRF at 6 months post-OLT was associated with pre-OLT renal dysfunction and 1 month post-OLT CRF. Four patients died in the ARF group, whereas 3 patients died in the group without ARF. We confirmed ARF to be a predictive event for short-term renal dysfunction. The majority of patients recovered renal function after the first month. Although many pre-, peri-, and post-OLT factors may contribute to the development of posttransplantation CRF, pre-OLT CRF seemed to be the most important risk factor.

Bone disorders in patients with chronic liver disease awaiting liver transplantation.

BACKGROUND: An important complication of chronic liver disease is osteodystrophy, which includes osteoporosis and the much rarer osteomalacia. Both conditions are associated with significant morbidity through fractures resulting in pain, deformity, and immobility. Liver transplantation may further deteriorate bone metabolism. The aim of the present study was to investigate the frequency and severity of hepatic osteodystrophy among patients with liver cirrhosis who were referred for liver transplantation. We also evaluated modifications in bone metabolism after liver transplantation. MATERIALS AND METHODS: We recruited 35 consecutive patients with chronic liver disease who were undergoing assessment for transplantation over a 1-year period. Bone mass in the total skeleton and proximal hip was evaluated using a dual-energy X-ray absorptiometry device (Lunar Prodigy Advance, GE Healthcare, USA). According to World Health Organization recommendations, osteoporosis was defined as a T score < -2.5 and osteopenia as T score between -1 and -2.5. RESULTS: We enrolled in the study 35 patients, including 8 females and 27 males of overall mean age of 57 +/- 7, who showed a viral etiology (57%) or alcohol etiology (28%), Child-Pugh 8.7 +/- 2.3. The overall prevalence of osteodystrophy was 40% (26% osteopenia and 14% osteoporosis). No difference was evident according to gender, severity of liver disease (Child-Pugh, Model for End-stage Liver Disease), or origin of liver disease. A subgroup of 10 transplanted patients reached 3-month follow-up, showing total body T score with a significant decrease after 3 months while femoral T scores tended to decrease insignificantly. CONCLUSIONS: This study revealed a high prevalence of low bone mineral density among cirrhotic patients before liver transplantation. We suggest that both bone mineral density and biochemical examinations should be considered to be routine tests to identify the status of bone mass and bone metabolism among recipients prior to liver transplantation.

Conversion from twice-daily to once-daily tacrolimus administration in liver transplant patient.

BACKGROUND: Compliance to immunosuppressive therapy is critical to prevent organ rejection and possible graft loss. A once-daily Tacrolimus formulation that may improve adherence-to-therapy while allowing the same patient care strategies, total daily dose and monitoring techniques that have been recently approved. The present study was sought to evaluate the feasibility of this formulation among liver transplantation patients (OLT). MATERIALS AND METHODS: Patients transplanted for at least 6 months were enrolled if they had stable doses of Tacrolimus over the last 3 months. Conversion from a twice to a once-daily regimen was based on a 1 mg:1 mg proportion. Tacrolimus blood levels were assessed at 0, 15, 30, 60, 90 days as well and 6 months after conversion. We recorded liver and renal function as well as adverse events. RESULTS: Among twenty-eight patients enrolled in the study including 23 males and 5 females the overall mean age was 59 +/- 8 years and the mean distance from OLT was 39 +/- 22. 32% of patients did not require any dose adjustment. In contrast, 43% required an increase (+0.6 +/- 0.3 mg/d), while 25%, a decrease (-0.5 +/- 0.0 mg/d) in the drug dose to maintain the same tacrolimus blood concentrations as at baseline. Ninety percent of patients stabilized blood levels within 45 days. None of the patients experienced adverse events or alterations in liver function. CONCLUSIONS: Our study confirmed that once-daily Tacrolimus is a useful therapeutic option for OLT patients; however dose adjustments are frequently needed in the short term. The drug is safe and may improve patient compliance.

Genetically detoxified mutants of heat-labile toxin from Escherichia coli are able to act as oral adjuvants.

Detoxified mutants of the Escherichia coli heat-labile toxin (LT) act as mucosal adjuvants to intranasally presented coadministered antigens. Here, we compare the adjuvant activity of a panel of detoxified derivatives of LT, using both intranasal (i.n.) and oral (p.o.) routes of administration. The mutants used as adjuvants varied in sensitivity to proteases and toxicity. With keyhole limpet hemocyanin (KLH) as the bystander antigen, the immune responses to i. n. immunizations were consistently higher than the equivalent p.o. -delivered proteins. LT-G192, a mutant which demonstrates a 10-fold reduction in toxicity in vitro, demonstrated wild-type adjuvant activity both i.n. and p.o., inducing similar titers of KLH specific antibody in the sera and immunoglobulin A in local mucosal secretions as wild-type LT. In line with previous data, the nontoxic holotoxoid LT-K63 induced intermediate immune responses in both the serum and mucosal secretions which were lower than those achieved with wild-type LT but at least 10-fold higher than those measured when the antigen was administered with LT-B. Although significant levels of local and systemic anti-KLH antibodies were induced following p.o. immunization with LT-K63, cellular proliferative responses to KLH was poor or undetectable. In contrast, LT and LT-G192 induced significant T-cell responses to KLH following p.o. immunization. These proliferating cells secreted both gamma interferon and interleukin-5, suggesting that the type of immune response induced following p.o. coimmunization with LT and purified protein is a mixed Th1/Th2 response.

Mucosal immunogenicity of genetically detoxified derivatives of heat labile toxin from Escherichia coli.

Using a fixed dose of antigen, the immune response to detoxified mutants of LT-WT following intranasal (i.n.), subcutaneous (s.c.) and oral (i.g.) immunisation has been studied. When given i.n., both LT-WT and mutant toxin, K63, generated significant levels of toxin-specific IgG in the serum, and the levels of IgA in nasal and lung lavages were greater than those induced by rLT-B. In comparison, i.g. immunisation of mice with a similar quantity of either LT-WT or K63 toxin induced barely detectable levels of IgG in the sera. However, if the amount of protein used for i.g. immunisation was increased tenfold, relatively good levels of toxin-specific IgG were induced in the sera by both LT-WT or K63. Low levels of toxin-specific IgA were also observed in intestinal washes from these mice. Western blotting of the sera, using the native toxin as an antigen, demonstrated the presence of both anti-A and anti-B subunit antibodies. Most significantly, toxin-neutralising antibodies were induced in the serum, with the strongest activity being induced by the LT-WT, an intermediate activity induced by mutant K63 and a lower response by rLT-B. Together, these data show that ADP-ribosyltransferase is not necessary for mucosal immunogenicity of these proteins, and that the i.n. route of immunisation is more effective than the i.g. route of immunisation for the generation of both systemic (IgG) and mucosal (IgA) immune responses.

Protection against Helicobacter pylori infection in mice by intragastric vaccination with H. pylori antigens is achieved using a non-toxic mutant of E. coli heat-labile enterotoxin (LT) as adjuvant.

We have previously shown that infection of mice with H. pylori can be prevented by oral immunization with H. pylori antigens given together with E. coli heat-labile enterotoxin (LT) as adjuvant. Since LT cannot be used in humans because of its unacceptable toxicity, we investigated whether protection of mice could be achieved by co-administration of antigens with non-toxic LT mutants. Here we show that CD1/SPF mice are protected against infection after oral vaccination with either purified H. pylori antigens (native and recombinant VacA, urease and CagA), or whole-cell vaccine formulations, given together with the non-toxic mutant LTK63 as a mucosal adjuvant. Furthermore we show that such protection is antigen-specific since immunization with recombinant or native VacA plus LTK63 conferred protection against infection by an H. pylori Type I strain, which expresses VacA, but not against challenge with a Type II strain which is not able to express this antigen. These results show that: (1) protection against H. pylori can be achieved in the mouse model of infection using subunit recombinant constructs plus non-toxic mucosal adjuvants; and (2) this mouse model is an useful tool in testing H. pylori vaccine formulations for eventual use in humans.

Mucosal adjuvanticity and immunogenicity of LTR72, a novel mutant of Escherichia coli heat-labile enterotoxin with partial knockout of ADP-ribosyltransferase activity.

Heat-labile Escherichia coli enterotoxin (LT) has the innate property of being a strong mucosal immunogen and adjuvant. In the attempt to reduce toxicity and maintain the useful immunological properties, several LT mutants have been produced. Some of these are promising mucosal adjuvants. However, so far, only those that were still toxic maintained full adjuvanticity. In this paper we describe a novel LT mutant with greatly reduced toxicity that maintains most of the adjuvanticity. The new mutant (LTR72), that contains a substitution Ala --> Arg in position 72 of the A subunit, showed only 0.6% of the LT enzymatic activity, was 100,000-fold less toxic than wild-type LT in Y1 cells in vitro, and was at least 20 times less effective than wild-type LT in the rabbit ileal loop assay in vivo. At a dose of 1 microg, LTR72 exhibited a mucosal adjuvanticity, similar to that observed with wild-type LT, better than that induced by the nontoxic, enzymatically inactive LTK63 mutant, and much greater than that of the recombinant B subunit. This trend was consistent for both the amounts and kinetics of the antibody induced, and priming of antigen-specific T lymphocytes. The data suggest that the innate high adjuvanticity of LT derives from the independent contribution of the nontoxic AB complex and the enzymatic activity. LTR72 optimizes the use of both properties: the enzymatic activity for which traces are enough, and the nontoxic AB complex, the effect of which is dose dependent. In fact, in dose-response experiments in mice, 20 microg of LTR72 were a stronger mucosal adjuvant than wild-type LT. This suggests that LTR72 may be an excellent candidate to be tested in clinical trials.

Protease susceptibility and toxicity of heat-labile enterotoxins with a mutation in the active site or in the protease-sensitive loop.

To generate nontoxic derivatives of Escherichia coli heat-labile enterotoxin (LT), site-directed mutagenesis has been used to change either the amino acid residues located in the catalytic site (M. Pizza, M. Domenighini, W. Hol, V. Giannelli, M. R. Fontana, M. M. Giuliani, C. Magagnoli, S. Peppoloni, R. Manetti, and R. Rappuoli, Mol. Microbiol. 14:51-60, 1994) or those located in the proteolytically sensitive loop that joins the A1 and A2 moieties of the A subunit (C. C. R. Grant, R. J. Messer, and W. J. Cieplack, Infect. Immun. 62:4270-4278, 1994; B. L. Dickinson and J. D. Clements, Infect. Immun. 63:1617-1623, 1995). In this work, we compared the in vitro and in vivo toxic properties and the resistance to protease digestion of the prototype molecules obtained by both approaches (LT-K63 and LT-R192G, respectively). As expected, LT-K63 was normally processed by proteases, while LT-R192G showed increased resistance to trypsin in vitro and was digested by trypsin only under denaturing conditions (3.5 M urea) or by intestinal proteases. No toxicity was detected with the LT-K63 mutant, even when 40 micrograms and 1 mg were used in the in vitro and in vivo assays, respectively. In marked contrast, LT-R192G showed only a modest (10-fold) reduction in toxicity in Y1 cells with a delay in the appearance of the toxic activity and had toxicity comparable to that of wild-type LT in the rabbit ileal loop assay. We conclude that mutagenesis of the active site generates molecules that are fully devoid of toxicity, while mutagenesis of the A1-A2 loop generates molecules that are resistant to trypsin in vitro but still susceptible to proteolytic activation by proteases other than trypsin, and therefore they may still be toxic in tissue culture and in vivo.

Mutations in the A subunit affect yield, stability, and protease sensitivity of nontoxic derivatives of heat-labile enterotoxin.

Heat-labile toxin (LT) is a protein related to cholera toxin, produced by enterotoxigenic Escherichia coli strains, that is organized as an AB5 complex. A number of nontoxic derivatives of LT, useful for new or improved vaccines against diarrheal diseases or as mucosal adjuvants, have been constructed by site-directed mutagenesis. Here we have studied the biochemical properties of the nontoxic mutants LT-K7 (Arg-7-->Lys), LT-D53 (Val-53-->Asp), LT-K63 (Ser-63-->Lys), LT-K97 (Val-97-->Lys), LT-K104 (Tyr-104-->Lys), LT-K114 (Ser-114-->Lys), and LT-K7/K97 (Arg-7-->Lys and Val-97-->Lys). We have found that mutations in the A subunit may have profound effects on the ability to form the AB5 structure and on the stability and trypsin sensitivity of the purified proteins. Unstable mutants, during long-term storage at 4 degrees C, showed a decrease in the amount of the assembled protein in solution and a parallel appearance of soluble monomeric B subunit. This finding suggests that the stability of the B pentamer is influenced by the A subunit which is associated with it. Among the seven nontoxic mutants tested, LT-K63 was found to be efficient in AB5 production, extremely stable during storage, resistant to proteolytic attack, and very immunogenic. In conclusion, LT-K63 is a good candidate for the development of antidiarrheal vaccines and mucosal adjuvants.

Acquired lymphangiectasis following mastectomy and radiation therapy--report of a case and review of the literature.

A woman noted vesicles and papules on her left upper extremity fifteen years after she underwent mastectomy and received radiation treatment for left breast carcinoma. The vesicles showed clinical and pathologic features of acquired lymphangiectasis. The skin lesions were successfully treated with shave excision followed by electrodesiccation and sequential peristaltic compressions twice daily.

Construction of nontoxic derivatives of cholera toxin and characterization of the immunological response against the A subunit.

Using computer modelling, we have identified some of the residues of the A subunit of cholera toxin (CT) and heat-labile toxin that are involved in NAD binding, catalysis, and toxicity. Here we describe the site-directed mutagenesis of the CT gene and the construction of CT mutants. Nine mutations of the A subunit gene were generated. Six of them encoded proteins that were fully assembled in the AB5 structure and were nontoxic; these proteins were CT-D53 (Val-53-->Asp), CT-K63 (Ser-63-->Lys), CT-K97 (Val-97-->Lys), CT-K104 (Tyr-104-->Lys), CT-S106 (Pro-106-->Ser), and the double mutant CT-D53/K63 (Val-53-->Asp, Ser-63-->Lys). Two of the mutations encoded proteins that were assembled into the AB5 structure but were still toxic; these proteins were CT-H54 (Arg-54-->His) and CT-N107 (His-107-->Asn). Finally, one of the mutant proteins, CT-E114 (Ser-114-->Glu), was unable to assemble the A and the B subunits and produced only the B oligomer. The six nontoxic mutants were purified from the culture supernatants of recombinant Vibrio cholerae strains and further characterized. The CT-K63 mutant, which was the most efficient in assembly of the AB5 structure, was used to immunize rabbits and was shown to be able to induce neutralizing antibodies against both the A and B subunits. This molecule may be useful for the construction of improved vaccines against cholera.

A genetically detoxified derivative of heat-labile Escherichia coli enterotoxin induces neutralizing antibodies against the A subunit.

Escherichia coli enterotoxin (LT) and the homologous cholera toxin (CT) are A-B toxins that cause travelers' diarrhea and cholera, respectively. So far, experimental live and killed vaccines against these diseases have been developed using only the nontoxic B portion of these toxins. The enzymatically active A subunit has not been used because it is responsible for the toxicity and it is reported to induce a negligible titer of toxin neutralizing antibodies. We used site-directed mutagenesis to inactivate the ADP-ribosyltransferase activity of the A subunit and obtained nontoxic derivatives of LT that elicited a good titer of neutralizing antibodies recognizing the A subunit. These LT mutants and equivalent mutants of CT may be used to improve live and killed vaccines against cholera and enterotoxinogenic E. coli.

Probing the structure-activity relationship of Escherichia coli LT-A by site-directed mutagenesis.

Computer analysis of the crystallographic structure of the A subunit of Escherichia coli heat-labile toxin (LT) was used to predict residues involved in NAD binding, catalysis and toxicity. Following site-directed mutagenesis, the mutants obtained could be divided into three groups. The first group contained fully assembled, non-toxic new molecules containing mutations of single amino acids such as Val-53-->Glu or Asp, Ser-63-->Lys, Val-97-->Lys, Tyr-104-->Lys or Asp, and Ser-114-->Lys or Glu. This group also included mutations in amino acids such as Arg-7, Glu-110 and Glu-112 that were already known to be important for enzymatic activity. The second group was formed by mutations that caused the collapse or prevented the assembly of the A subunit: Leu-41-->Phe, Ala-45-->Tyr or Glu, Val-53-->Tyr, Val-60-->Gly, Ser-68-->Pro, His-70-->Pro, Val-97-->Tyr and Ser-114-->Tyr. The third group contained those molecules that maintained a wild-type level of toxicity in spite of the mutations introduced: Arg-54-->Lys or Ala, Tyr-59-->Met, Ser-68-->Lys, Ala-72-->Arg, His or Asp and Arg-192-->Asn. The results provide a further understanding of the structure-function of the active site and new, non-toxic mutants that may be useful for the development of vaccines against diarrhoeal diseases.