The spectrum of thrombotic microangiopathy related to monoclonal gammopathy.

Background: Recent studies showed a high prevalence of monoclonal gammopathy (MG) in patients with thrombotic microangiopathy (TMA) aged over 50 years and suggested that complement dysregulation is pivotal for the disease to develop. Here, we studied this premise in seven patients with TMA and coexisting MG. Methods: Patients with TMA on kidney biopsy and/or peripheral blood were recruited from the prospective COMPETE cohort (NCT04745195) and Limburg Renal Registry. Patients were screened for complement dysregulation, including genetics/factor H autoantibodies (FHAA) and functional ex vivo testing on microvascular endothelial cells. Results: Seven (8%) out of 84 patients with TMA presented with a coexisting MG. MG clustered in patients aged over 50 years (n/N = 6/32, 19%). C4 and/or C3 levels were low in three patients, while four patients presented with normal complement levels. None of the patients carried rare variants in complement genes. Massive ex vivo C5b9 formation on the endothelium was noted in one patient; purified IgG from this patient caused massive ex vivo C5b9 formation via the alternative pathway of complement activation, pointing to complement dysregulation in the fluid phase. Kidney biopsies from other nephropathies linked to MG rarely exhibited concurrent TMA (n/N = 1/27, 4%). Conclusions: MG clustered in patients with TMA aged over 50 years. TMA and coexisting MG represents a heterogeneous disease spectrum, including a small subset of patients who may present with complement dysregulation.

Effect of small-quantity lipid-based nutrient supplements on growth, psychomotor development, iron status, and morbidity among 6- to 12-mo-old infants in South Africa: a randomized controlled trial.

Background: Evidence on the effect of small-quantity lipid-based nutrient supplements (SQ-LNSs) on early child growth and development is mixed. Objective: This study assessed the effect of daily consumption of 2 different SQ-LNS formulations on linear growth (primary outcome), psychomotor development, iron status (secondary outcomes), and morbidity in infants from age 6 to 12 mo within the context of a maize-based complementary diet. Methods: Infants (n = 750) were randomly assigned to receive SQ-LNS, SQ-LNS-plus, or no supplement. Both SQ-LNS products contained micronutrients and essential fatty acids. SQ-LNS-plus contained, in addition, docosahexaenoic acid, arachidonic acid (important for brain and eye development), lysine (limiting amino acid in maize), phytase (enhances iron absorption), and other nutrients. Infants' weight and length were measured bimonthly. At age 6 and 12 mo, psychomotor development using the Kilifi Developmental Inventory and South African Parent Rating Scale and hemoglobin, plasma ferritin, C-reactive protein, and α1-acid glycoprotein were assessed. WHO Motor Milestone outcomes, adherence, and morbidity were monitored weekly through home visits. Primary analysis was by intention-to-treat, comparing each SQ-LNS group with the control. Results: SQ-LNS-plus had a positive effect on length-for-age zscore at age 8 mo (mean difference: 0.11; 95% CI: 0.01, 0.22; P = 0.032) and 10 mo (0.16; 95% CI: 0.04, 0.27; P = 0.008) but not at 12 mo (0.09; 95% CI: -0.02, 0.21; P = 0.115), locomotor development score (2.05; 95% CI: 0.72, 3.38; P = 0.003), and Parent Rating Score (1.10; 95% CI: 0.14, 2.07; P = 0.025), but no effect for weight-for-age zscore. Both SQ-LNS (P = 0.027) and SQ-LNS-plus (P = 0.005) improved hemoglobin concentration and reduced the risk of anemia, iron deficiency, and iron-deficiency anemia. Both SQ-LNS products reduced longitudinal prevalence of fever, coughing, and wheezing but increased incidence and longitudinal prevalence of diarrhea, vomiting, and rash/sores. Conclusions: Point-of-use fortification with SQ-LNS-plus showed an early transient effect on linear growth and improved locomotor development. Both SQ-LNS products had positive impacts on anemia and iron status. This trial was registered at clinicaltrials.gov as NCT01845610.

Inhibition of iron absorption by calcium is modest in an iron-fortified, casein- and whey-based drink in Indian children and is easily compensated for by addition of ascorbic acid.

BACKGROUND: Calcium inhibits and ascorbic acid (AA) enhances iron absorption from iron-fortified foods. Absorption efficiency depends on iron status, although the interaction is unclear. OBJECTIVE: We investigated the ability of AA to overcome calcium-induced inhibition of iron absorption in children differing in iron status. METHODS: The effect of calcium (0, 100, and 200 mg/test meal) on iron absorption in the absence and presence of AA (0, 42.5, and 85 mg/test meal) from a casein/whey-based drink fortified with ferrous sulfate was assessed in a series of randomized crossover studies both in iron-replete (IR) Indian schoolchildren and in children with iron deficiency anemia (IDA) (6-11 y; n = 14-16/group) by using stable isotopes. RESULTS: In the absence of calcium and AA, iron absorption from the casein/whey-based drink was 20% lower in IR children than in children with IDA. The addition of calcium reduced mean iron absorption by 18-27%, with the effect being stronger for high added calcium (P < 0.01). AA at a 2:1 or 4:1 molar ratio enhanced iron absorption by a factor of 2-4 and greatly overcompensated for the inhibitory effect of calcium on iron absorption in a dose-dependent manner (P < 0.001). The dose-response effect tended to be stronger (P < 0.1) in the IDA group, and iron status was of far less influence on iron absorption than the enhancing effect of AA. CONCLUSION: When adding AA to iron-fortified milk products, care should be taken not to provide absorbable iron in excess of needs.

Assessment of inflammatory resilience in healthy subjects using dietary lipid and glucose challenges.

BACKGROUND: Resilience or the ability of our body to cope with daily-life challenges has been proposed as a new definition of health, with restoration of homeostasis as target resultant of various physiological stress responses. Challenge models may thus be a sensitive measure to study the body's health. The objective of this study was to select a dietary challenge model for the assessment of inflammatory resilience. Meals are a challenge to metabolic homeostasis and are suggested to affect inflammatory pathways, yet data in literature are limited and inconsistent. METHOD: The kinetic responses of three different dietary challenges and a water control challenge were assessed on various metabolic and inflammatory markers in 14 healthy males and females using a full cross-over study design. The dietary challenges included glucose (75 g glucose in 300 ml water), lipids (200 ml whipping cream) and a mix of glucose and lipids (same amounts as above), respectively. Blood samples were collected at baseline and at 0.5, 1, 2, 4, 6, 8 and 10 h after consumption of the treatment products. Inflammation (IFNγ, IL-1ß, IL-6, IL-8, IL-10, IL-12p70, TNF-α CRP, ICAM-1, VCAM-1, SAA, E-selectin, P-selectin, thrombomodulin, leukocytes, neutrophils, lymphocytes) and clinical (e.g. glucose, insulin, triglycerides) markers as well as gene expression in blood cells and plasma oxylipin profiles were measured. RESULTS: All three dietary challenges induced changes related to metabolic control such as increases in glucose and insulin after the glucose challenge and increases in triglycerides after the lipid challenge. In addition, differences between the challenges were observed for precursor oxylipins and some downstream metabolites including DiHETrE's and HODE's. However, none of the dietary challenges induced an acute inflammatory response, except for a modest increase in circulating leukocyte numbers after the glucose and mix challenges. Furthermore, subtle, yet statistically significant increases in vascular inflammatory markers (sICAM-1 and sVCAM-1) were found after the mix challenge, when compared to the water control challenge. CONCLUSIONS: This study shows that dietary glucose and lipid challenges did not induce a strong acute inflammatory response in healthy subjects, as quantified by an accurate and broad panel of parameters.

Rice-based oral antibody fragment prophylaxis and therapy against rotavirus infection.

Rotavirus-induced diarrhea is a life-threatening disease in immunocompromised individuals and in children in developing countries. We have developed a system for prophylaxis and therapy against rotavirus disease using transgenic rice expressing the neutralizing variable domain of a rotavirus-specific llama heavy-chain antibody fragment (MucoRice-ARP1). MucoRice-ARP1 was produced at high levels in rice seeds using an overexpression system and RNAi technology to suppress the production of major rice endogenous storage proteins. Orally administered MucoRice-ARP1 markedly decreased the viral load in immunocompetent and immunodeficient mice. The antibody retained in vitro neutralizing activity after long-term storage (>1 yr) and boiling and conferred protection in mice even after heat treatment at 94°C for 30 minutes. High-yield, water-soluble, and purification-free MucoRice-ARP1 thus forms the basis for orally administered prophylaxis and therapy against rotavirus infections.

Anti-rotavirus protein reduces stool output in infants with diarrhea: a randomized placebo-controlled trial.

BACKGROUND & AIMS: Rotavirus infection is a leading cause of morbidity and mortality in children younger than 5 years of age. Current treatment options are limited. We assessed the efficacy of a llama-derived, heavy-chain antibody fragment called anti-rotavirus protein (ARP1), in modifying the severity and duration of diarrhea in male infants with rotavirus infection. METHODS: We performed a double-blind, placebo-controlled trial of 176 male infants (6-24 months old) with severe rotavirus-associated diarrhea at Dhaka Hospital, Bangladesh. The infants were randomly assigned to groups given oral ARP1 (15-30 mg/kg/day, n = 88) or placebo (maltodextrin, n = 88) for a maximum of 5 days. The primary outcomes were severity (stool output) and duration of diarrhea and fecal excretion of rotavirus. Secondary outcomes were intake of oral rehydration salt solution, severity of vomiting, and serum levels of rotavirus-specific IgA. RESULTS: In infants with only rotavirus infection, total cumulative stool output was 305.47 g/kg body weight among those given placebo (n = 63) and 237.03 g/kg body weight among those given ARP1 (n = 61) (a difference of 68.44 g/kg body weight or 22.5%; 95% confidence interval: 18.27-118.59 g/kg body weight; P =.0079). There was a significant reduction in rate of stool output (g/kg/d) in the ARP1 group compared with the placebo group (61%; P = .002). ARP1 had no significant effect in infants with concomitant infections or on any other measured outcomes. No adverse events could be linked to ARP1. CONCLUSIONS: In a placebo-controlled trial, ARP1 reduced stool output in male infants with severe rotavirus-associated diarrhea. Clinicaltrials.gov number: NCT01259765.

Insight into advance care planning for patients on dialysis.

CONTEXT: Advance care planning is not included in regular clinical care for patients on dialysis. Insight into life-sustaining treatment preferences and communication about end-of-life care is necessary to develop interventions to improve advance care planning for patients on dialysis. OBJECTIVES: This cross-sectional observational study aimed to understand the preferences for life-sustaining treatments of outpatients on dialysis and to study the quality of patient-physician communication about end-of-life care and barriers and facilitators to this communication. METHODS: The following outcomes were assessed in 80 clinically stable dialysis patients: demographics, clinical characteristics, life-sustaining treatment preferences (cardiopulmonary resuscitation and mechanical ventilation, and Willingness to Accept Life-Sustaining Treatment instrument), preference for site of death, quality of communication (Quality of Communication Questionnaire), and barriers and facilitators to communication about end-of-life care (Barriers and Facilitators Questionnaire). RESULTS: Patients were able to indicate their preferences for life-sustaining treatments and site of death. Preferences for life-sustaining treatments depend on the specific treatment, the expected outcome of treatment, and likelihood of an adverse outcome. Life-sustaining preferences were discussed with the nephrologist by 30.3% of the patients. Quality of the patient-physician communication about end-of-life care was rated poor. This study identified several barriers and facilitators to end-of-life care communication. CONCLUSION: Patients should receive information about treatment burden, expected outcome, and the likelihood of an adverse outcome when discussing life-sustaining treatments. Quality of patient-physician communication about end-of-life care needs to improve. Barriers and facilitators to communication about end-of-life care provide direction for future interventions to facilitate advance care planning for patients on dialysis.

In vitro neutralisation of rotavirus infection by two broadly specific recombinant monovalent llama-derived antibody fragments.

Rotavirus is the main cause of viral gastroenteritis in young children. Therefore, the development of inexpensive antiviral products for the prevention and/or treatment of rotavirus disease remains a priority. Previously we have shown that a recombinant monovalent antibody fragment (referred to as Anti-Rotavirus Proteins or ARP1) derived from a heavy chain antibody of a llama immunised with rotavirus was able to neutralise rotavirus infection in a mouse model system. In the present work we investigated the specificity and neutralising activity of two llama antibody fragments, ARP1 and ARP3, against 13 cell culture adapted rotavirus strains of diverse genotypes. In addition, immunocapture electron microscopy (IEM) was performed to determine binding of ARP1 to clinical isolates and cell culture adapted strains. ARP1 and ARP3 were able to neutralise a broad variety of rotavirus serotypes/genotypes in vitro, and in addition, IEM showed specific binding to a variety of cell adapted strains as well as strains from clinical specimens. These results indicated that these molecules could potentially be used as immunoprophylactic and/or immunotherapeutic products for the prevention and/or treatment of infection of a broad range of clinically relevant rotavirus strains.

Simultaneous blood temperature control and blood volume control reduces intradialytic symptoms.

PURPOSE: Intra-dialytic morbid events (IME; e.g. hypotension, cramps, headaches) are frequent complications during hemodialysis (HD), known to be associated with ultrafiltration-induced hypovolemia and body temperature changes. Feedback control of blood volume adjusts the ultrafiltration rate in order to keep the blood volume above the patient's individual limit; feedback control of blood temperature maintains the mean arterial blood temperature at the individual pre-dialytic level. Each of these methods reduces the frequency of IME. METHODS: In a randomized clinical trial the simultaneous application of both feedback controls was investigated for the first time. In 15 weeks, each patient went through 3 study phases: an observational screening phase, a standard phase (STD), and a blood temperature- and blood volume-control phase (CTL). Patients with at least 5 sessions with IME out of 15 sessions in the screening phase were eligible for the study and randomized either into sequence STD-CTL or CTL-STD. RESULTS: 26 patients completed the study according to protocol, and 778 HD treatments were analyzed. The general treatment parameters were similar in both study phases: treatment duration (STD: 244 min, CTL: 243 min, NS), pre-dialytic weight (STD: 72.3 kg, CTL: 72.2 kg, NS), and weight loss due to ultrafiltration (STD: 3.26 kg, CTL: 3.15 kg, NS). The proportion of HD treatments with IME was 32.8% during STD and 18.0% during CTL (p=0.024). CONCLUSIONS: The frequency of HD sessions with IME was significantly reduced by 45% compared to standard HD in this randomized clinical trial by use of individualized HD treatments with simultaneous feedback control of blood volume and blood temperature.

Lactobacilli producing bispecific llama-derived anti-rotavirus proteins in vivo for rotavirus-induced diarrhea.

AIMS: Using genetically engineered lactobacilli, producing high avidity llama VHH domains (referred to as anti-rotavirus proteins; ARPs), to test the effect of multimeric antibody fragments as prophylaxis and therapy against rotavirus infection. METHODS: Two ARPs, ARP1 and ARP3, shown to bind to different epitopes and act synergistically against rotavirus, were displayed on the surface of Lactobacillus paracasei as monovalent or bivalent proteins (mono- or bi-specific). RESULTS: Although a nonsignificant difference was observed between lactobacilli producing bispecific ARP3-ARP1 and monomeric ARPs, lactobacilli producing bispecific ARP3-ARP1 were superior at reducing the rate of diarrhea when used for prophylactic and therapeutic intervention in a mouse model of rotavirus infection in comparison to nontreated animals. CONCLUSION: Expression of bispecific antibodies in lactobacilli resulted in slight improvement of their efficacy. Furthermore, increasing the specificity would theoretically reduce the rate of appearance of viral escape mutants and would have a broader capacity to be effective against a range of viral serotypes.

Random serial sampling to evaluate efficacy of iron fortification: a randomized controlled trial of margarine fortification with ferric pyrophosphate or sodium iron edetate.

BACKGROUND: Random serial sampling is widely used in population pharmacokinetic studies and may have advantages compared with conventional fixed time-point evaluation of iron fortification. OBJECTIVE: Our objective was to validate random serial sampling to judge the efficacy of iron fortification of a low-fat margarine. DESIGN: We conducted a 32-wk placebo-controlled, double-blind, iron-intervention trial in 18-40-y-old Swiss women (n = 142) with serum ferritin (SF) concentrations <25 µg/L. Women were randomly assigned to 3 groups to receive 20 g margarine, with 14 mg added iron as either micronized ground ferric pyrophosphate (MGFePP) or sodium iron edetate (NaFeEDTA), or placebo daily. We measured hemoglobin and iron status of subjects at 2 fixed time points (at baseline and the endpoint) plus 3 randomly assigned time points between 4 and 28 wk. With the use of bootstrapping, the number of observations per individual was reduced to 3 and then compared with the 5-time-point data. Mixed-effects models were used to estimate iron repletion over time for random sampling, and analysis of covariance was used for fixed time-point sampling. RESULTS: Body iron stores increased in women who received MGFePP or NaFeEDTA compared with women who received placebo (P < 0.05). The increase in body iron stores with NaFeEDTA fortification was 2-3 times the increase with MGFePP fortification (P < 0.05); the difference was more marked in women with baseline SF concentrations <15 µg/L (P < 0.05). Random serial sampling reduced the required sample size per group to one-tenth of that for 2 fixed time points. Compared with the 5-time-point analysis, the 3-time-point sparse sampling generated comparable estimates of efficacy. CONCLUSIONS: When used to evaluate the efficacy of iron fortificants, random serial sampling can reduce the sample size, invasiveness, and costs while increasing sensitivity. Random serial sampling more clearly describes the pattern of iron repletion and may prove useful in evaluating other micronutrient interventions.

In vitro bioavailability of iron from the heme analogue sodium iron chlorophyllin.

The use of heme analogues from vegetable origin could provide an alternative iron source of potentially high bioavailability. Sodium iron chlorophyllin is a water-soluble semisynthetic chlorophyll derivative where the magnesium in the porphyrin ring has been substituted by iron. We have used an in vitro model that combines gastric and intestinal digestion followed by intestinal iron uptake in Caco-2 cells to determine the bioavailability of iron from sodium iron chlorophyllin. Our results demonstrate that sodium iron chlorophyllin is stable under simulated gastrointestinal conditions and is able to deliver bioavailable iron to Caco-2 cells. Similar to the heme, the bioavailability of iron from sodium iron chlorophyllin is dependent on the food matrix, and it was inhibited by calcium. Potentially, sodium iron chlorophyllin could be used as an iron fortificant from vegetable origin with high bioavailability.

An evaluation of blood volume changes during ultrafiltration pulses and natriuretic peptides in the assessment of dry weight in hemodialysis patients.

Changes in blood volume (BV) during dialysis as well as plasma levels of brain natriuretic peptide (BNP) and N-terminal (NT) pro-BNP levels are possible tools to assess dry weight in hemodialysis (HD) patients. The aim of the study was to compare these parameters with other non-invasive techniques used to assess dry weight in HD patients, and to study their relation with intradialytic hypotension (IDH) and the presence of cardiovascular disease BV changes during HD, both during regular dialysis and during an ultrafiltration pulse, plasma levels of NT pro-BNP and BNP, and vena cava diameter index (VCDI) were assessed in a cohort of 66 HD patients, which was subdivided according to tertiles of total body water (TBW) corrected for body weight, assessed by bioimpedance analysis. Parameters were also related to the presence of IDH and history of cardiovascular disease. The decline in BV during regular dialysis and during an ultrafiltration pulse, as well as VCDI and BNP were significantly different between the tertiles of normalized TBW, but refill after the ultrafiltration pulse and NT pro-BNP were not. Only VCDI and the decline in BV during regular dialysis were significantly different between patients with or without IDH. Vena cava diameter index, BNP, and NT pro-BNP were significantly higher in patients with cardiovascular disease. Using bioimpedance as the reference method, changes in BV, either during regular dialysis or during an ultrafiltration pulse, as well as VCDI and BNP are all indicative of hydration state in dialysis patients, but refill after an ultrafiltration pulse is not. Only VCDI and BV changes were related to IDH. The presence of cardiovascular disease appears to influence both VCDI as well as BNP.

Lactobacilli expressing variable domain of llama heavy-chain antibody fragments (lactobodies) confer protection against rotavirus-induced diarrhea.

BACKGROUND: Rotavirus-induced diarrhea poses a worldwide medical problem in causing substantial morbidity and mortality among children in developing countries. We therefore developed a system for passive immunotherapy in which recombinant lactobacilli constitutively express neutralizing variable domain of llama heavy-chain (VHH) antibody fragments against rotavirus. METHODS: VHH were expressed in Lactobacillus paracasei, in both secreted and cell surface-anchored forms. Electron microscopy was used to investigate the binding efficacy of VHH-expressing lactobacilli. To investigate the in vivo function of VHH-expressing lactobacilli, a mouse pup model of rotavirus infection was used. RESULTS: Efficient binding of the VHH antibody fragments to rotavirus was shown by enzyme-linked immunosorbent assay and scanning electron microscopy. VHH fragments expressed by lactobacilli conferred a significant reduction in infection in cell cultures. When administered orally, lactobacilli-producing surface-expressed VHH markedly shortened disease duration, severity, and viral load in a mouse model of rotavirus-induced diarrhea when administered both fresh and in a freeze-dried form. CONCLUSIONS: Transformed lactobacilli may form the basis of a novel form of prophylactic treatment against rotavirus infections and other diarrheal diseases.

Therapeutic effect of llama derived VHH fragments against Streptococcus mutans on the development of dental caries.

Streptococcus mutans is the main cause of dental caries. We evaluated the therapeutic effect of variable regions of a llama heavy chain antibody fragments directed against S. mutans named S36-VHH (S for Streptococcus) alone or fused with glucose oxidase (GOx) from Aspergillus niger. Western blot analysis and ELISA revealed binding of the S36-VHH to the streptococcal antigen I/II adhesin molecule of S. mutans serotype C. In a rat-desalivated caries model, daily administration of S36-VHH significantly reduced the development of smooth surface caries. No additional therapeutic effect of GOx was observed. Our results suggest that llama VHH antibodies may be a potential benefit as prophylaxis against dental caries.

Llama antibodies against a lactococcal protein located at the tip of the phage tail prevent phage infection.

Bacteriophage p2 belongs to the most prevalent lactococcal phage group (936) responsible for considerable losses in industrial production of cheese. Immunization of a llama with bacteriophage p2 led to higher titers of neutralizing heavy-chain antibodies (i.e., devoid of light chains) than of the classical type of immunoglobulins. A panel of p2-specific single-domain antibody fragments was obtained using phage display technology, from which a group of potent neutralizing antibodies were identified. The antigen bound by these antibodies was identified as a protein with a molecular mass of 30 kDa, homologous to open reading frame 18 (ORF18) of phage sk1, another 936-like phage for which the complete genomic sequence is available. By the use of immunoelectron microscopy, the protein is located at the tip of the tail of the phage particle. The addition of purified ORF18 protein to a bacterial culture suppressed phage infection. This result and the inhibition of cell lysis by anti-ORF18 protein antibodies support the conclusion that the ORF18 protein plays a crucial role in the interaction of bacteriophage p2 with the surface receptors of Lactococcus lactis.

Induced refolding of a temperature denatured llama heavy-chain antibody fragment by its antigen.

In a previous study we have shown that llama VHH antibody fragments are able to bind their antigen after a heat shock of 90 degrees C, in contrast to the murine monoclonal antibodies. However, the molecular mechanism by which antibody:antigen interaction occurs under these extreme conditions remains unclear. To examine in more detail the structural and thermodynamic aspects of the binding mechanism, an extensive CD, ITC, and NMR study was initiated. In this study the interaction between the llama VHH -R2 fragment and its antigen, the dye Reactive Red-6 (RR6) has been explored. The data show clearly that most of the VHH-R2 population at 80 degrees C is in an unfolded conformation. In contrast, CD spectra representing the complex between VHH-R2 and the dye remained the same up to 80 degrees C. Interestingly, addition of the dye to the denatured VHH-R2 at 80 degrees C yielded the spectrum of the native complex. These results suggest an induced refolding of denatured VHH-R2 by its antigen under these extreme conditions. This induced refolding showed some similarities with the well established "induced fit" mechanism of antibody-antigen interactions at ambient temperature. However, the main difference with the "induced fit" mechanism is that at the start of the addition of the antigen most of the VHH molecules are in an unfolded conformation. The refolding capability under these extreme conditions and the stable complex formation make VHHs useful in a wide variety of applications.

Isolation of llama antibody fragments for prevention of dandruff by phage display in shampoo.

As part of research exploring the feasibility of using antibody fragments to inhibit the growth of organisms implicated in dandruff, we isolated antibody fragments that bind to a cell surface protein of Malassezia furfur in the presence of shampoo. We found that phage display of llama single-domain antibody fragments (VHHs) can be extended to very harsh conditions, such as the presence of shampoo containing nonionic and anionic surfactants. We selected several VHHs that bind to the cell wall protein Malf1 of M. furfur, a fungus implicated in causing dandruff. In addition to high stability in the presence of shampoo, these VHHs are also stable under other denaturing conditions, such as high urea concentrations. Many of the stable VHHs were found to contain arginine at position 44. Replacement of the native amino acid at position 44 with arginine in the most stable VHH that lacked this arginine resulted in a dramatic further increase in the stability. The combination of the unique properties of VHHs together with applied phage display and protein engineering is a powerful method for obtaining highly stable VHHs that can be used in a wide range of applications.