Accuracy of retrospective pain measurement in patients with chronic pain.

The use of pain scales that refer to a past time period is thereby based on the assumption that patients accurately remember their 'average', 'greatest' and 'least' pain. The present study addresses the validity of numerical pain rating scales that refer to a past period of time (herein, the past 7 days). Routine data from 94 patients with chronic pain were retrospectively analysed. Pain questionnaire data on the greatest, least and average pain during the past week and on current pain were compared with the mean value of entries in a pain diary from the corresponding period. The retrospectively assessed average, greatest and least pain values were consistently slightly higher than the corresponding values of daily current pain measured for the studied collective of chronic pain patients. Current pain (at the time of answering the questionnaire) better represents daily currently measured pain [intraclass correlation (ICC)=0.885] than retrospective individual measurements. The greatest correlation with averaged diary data was shown by the combination of questionnaire data on average, least and current pain (ICC=0.911). The high correlations between the questionnaire and diary data support the validity of retrospective pain surveys. However, the current status influences recall. Thus, composite retrospective pain data improve with the addition of current pain.

Conservative In-Patient Treatment of Specific Back Pain Before and During the Coronavirus Disease Pandemic. / Konservative stationäre Behandlung von spezifischen Rückenschmerzen vor und während der Corona-Pandemie.

BACKGROUND: The measures taken in the coronavirus disease pandemic have had major structural and financial effects on orthopaedics and trauma surgery as many surgeries in this field. Experience Appropriate reports from non-surgical orthopaedics are not yet available. AIMS OF THE STUDY: The study aimed to provide information on the changes that occurred in a group of patients with spinal diseases undergoing conservative in-patient treatment during the coronavirus disease pandemic, with regard to the number of cases, patient structure and course of treatment. MATERIALS AND METHODS: Data from a total of 954 patients from an acute department for conservative treatment of back pain from the years 2019 and 2020 were retrospectively compared, thus allowing conclusions to be drawn about the course of in-patient conservative spinal treatment. In addition to sociodemographic data, numerical pain rating scales, scales for impairment by pain and physical function at the beginning and at the end of treatment were analysed using descriptive statistics and differentiation tests. RESULTS: The study showed a 21% reduction in the number of cases in 2020 compared with those in 2019. The patient structure has changed in terms of diagnosis groups and physical function. The values of the assessments on discharge and their pre-post differences show an almost identical pattern of treatment outcomes before and after the start of the pandemic. CONCLUSIONS: The relatively small decline in the number of in-patient admissions for the non-surgical treatment of specific spinal disorders indicates that this treatment option was also necessary in the pandemic-related crisis. With minor changes in the patient structure, comparable treatment results could be achieved.

Auditing Practices of the Medical Service Regarding Hospitalizations of Patients With a Secondary Diagnosis of Obesity.

BACKGROUND: It has been observed that the Medical Service (Medizinischer Dienst, an auditing body of the German statutory health insurance system) is more likely to audit the bill for a hospitalization in a psychosomatic clinic if the patient carries a secondary diagnosis of obesity. METHODS: In an exploratory study, we retrospectively analyzed 771 datasets collected in 2019 as part of the standard documentation of acute psychosomatic hospitalizations. RESULTS: In 2019, the Medical Service audited bills for psychosomatic hospital - izations much more often in obese than in non-obese patients (odds ratio [OR] 2.499; 95% confidence interval [1.69; 3.69]). This was accounted for by a very high audit rate for patients with a secondary diagnosis of grade 3 obesity (OR = 3.972 [2.30; 6.86]). The audit categories "quality of coding" and "possible incorrect admission" were examined. CONCLUSION: Treatments of markedly obese inpatients that incurred greater expenses presumably led to a higher hospitalization audit rate as an automatic consequence of the auditing algorithms used. An unintentional statistical discrimination arose from the unjustified linkage of the audit category "quality of coding" of the secondary diagnosis (obesity) with the audit category "possible incorrect admission" with regard to the main diagnosis. Similar effects may be occurring with economically relevant secondary diagnoses in other areas of medicine as well.

Clinical Relevance of Changes in Pain Intensity in Patients with Specific Back Pain. / Klinische Bedeutung von Veränderungen der Schmerzstärke bei Patienten mit spezifischen Rückenschmerzen.

BACKGROUND: Pain intensity is frequently measured on the 11-point numerical pain rating scale (NRS-PI), ranging from 0 (no pain) to 10 points (worst imaginable pain). However, it is difficult to interpret the clinical importance of changes from baseline to endpoint on this instrument. OBJECTIVES: To estimate the minimal detectable change (MDC) and the minimal clinically important difference (MCID) for average pain intensity in patients with specific back pain. MATERIALS AND METHODS: Data on 1232 subjects with specific back pain from a German hospital were included in this study. A score combining the patient's (PGIC) and the physician's global impression of change (CGIC) over the in-patient length of stay was used as an external criterion. A priori, we considered the score value "slightly improved" as the MCID. MDC was calculated using the standard error of measurement (SEM) and the standard deviation (SD) of the sample. MCID was estimated by the mean value of PI-NRS change in patients who self-assess as "slightly improved", and by sensitivity/specificity analyses, computed by the receiver operating characteristic method (ROC). RESULTS: MDC was 1.77. The MCS and ROC methods consistently showed an MCID of 2 for the total sample. Both methods showed the dependence of the MCID on the initial pain: 1 for mild to moderate pain at baseline (1 - 4 NRS points), 2 for moderate to severe pain (5 - 7) and 3 - 4 for very severe to extreme pain (8 - 10). For patients with lumbosacral intervertebral disc disorders and patients in the acute phase (duration of pain < 6 weeks), the ROC method resulted in a higher limit than the MCS method. CONCLUSIONS: In order to facilitate the interpretation of changes and to take into account the patient's perspective, the global assessment of the success of treatment should be used as an anchor criterion. In addition to dealing with pain measurement, function-related and psychosocial aspects of pain symptoms should be kept in mind.

Ascertaining minimal clinically meaningful changes in symptoms of depression rated by the 15-item Centre for Epidemiologic Studies Depression Scale.

RATIONALE, AIMS AND OBJECTIVES: In clinical practise and in clinical studies on depression it is important to estimate whether changes in symptomatology measured by self-rating instruments are, in fact, clinically relevant. Therefore, the aim of the study was to estimate the clinical relevance of changes on the 15-item version of the Centre for Epidemiologic Studies Depression Scale (CES-D-15) based on the concept of the minimal clinically important difference (MCID). METHODS: Data was acquired from 4781 patients with depression symptoms from a German psychosomatic hospital who have been assessed using the CES-D-15 before and after treatment. Threshold values representing the MCID were estimated on the basis of mean change scores and sensitivity/specificity analyses. Patients' global impression of change, clinical (therapists') global impression of change and change in impairment severity were used as external anchor criteria. RESULTS: On average, the MCID was represented by a reduction of approximately 11 points in the CES-D-15, irrespective of age, gender, type of treatment and first or secondary diagnosis. However, higher baseline scores in the CES-D-15 required larger changes of raw values to represent a clinically important difference. CONCLUSIONS: Anchor-based values are suggested here as an estimation of the clinical relevance of changes in the CES-D-15. Thus, instead of relying solely on effect sizes, the evaluation of treatment outcomes should be supplemented by reporting the percentage of patients who have reached the MCID. Further examinations to verify our results in other patient populations and with other types of anchor criteria will be needed.

[Evaluation of Inpatient Conservative Management of Acute, Subacute, and Chronic Back Pain]. / Evaluation einer stationären nicht operativen Behandlung von akuten, subakuten und chronischen Rückenschmerzen.

BACKGROUND: Backache is very common in the German population. It is a common reason for people to seek medical advice and specific back pain programs have been developed. The intention of this study is to evaluate the short and long term effects of conservative management of back pain in a German general hospital. Outcomes of interest were pain intensity, interference in daily functions, physical functioning and health related quality of life. PATIENTS AND METHODS: We examined 1010 patients with acute, subacute and chronic back pain, admitted to inpatient nonsurgical interventional therapy in a German hospital between July 2013 and July 2015. Outcomes were assessed at the end of the inpatient treatment and at 3, 6 and 12 months follow-up, using Numerical Rating Scales (NRS) for pain and daily function, the Hannover Ability Questionnaire for Measuring Back Pain-Related Functional Limitations (FFbH - R) and the German Version of the EuroQol Questionnaire (EQ-5D) for measuring health-related quality of life. The baseline questionnaire also included questions on the risk of developing long-term disability following back pain (HKF-R 10) for acute and subacute cases and the Mainz Pain Staging System (MPSS) for patients with chronic back pain. The return rate was 54% after 3 months, 38% after 6 months and 27% at 12 months follow-up. The results from the follow-up measurements (T2 - T5) were compared to the pre-treatment results (T1). Because of missing or insufficiently normal distributions nonparametric paired Wilcoxon tests were used to test differences over time for each variable. Level of significance was adjusted for multiple testing. In addition, effect sizes were computed to estimate the clinical relevance of statistically significant results. RESULTS: Pain intensity and impact of pain on daily function were significantly lower at the end of the inpatient treatment (T2). The results remained largely stable at the 3, 6 and 12 month follow-ups. Significant improvements were found in physical functioning and health-related quality of life. These improvements were maintained equally at the 3, 6 and 12 month follow-ups. In consequence, working ability increased during the follow-up period. Calculated effect sizes showed large effects for pain intensity, interference and quality of life (r = 0.51 to 0.85) and predominately moderate effects (r = 0.45 to 0.62) for physical functioning at all measurement points. The percentage of patients who had an operation due to continuing back pain after conservative treatment was 7.8, 9.9, and 12.3 at the 3, 6, and 12 month follow-ups, respectively. CONCLUSION: Persistent effects of inpatient conservative treatment of back pain were found for all outcome variables. The specific approach appears to be effective in conservative treatment programs of back pain. In the end, it's not about the alternative of surgery or conservative treatment for back pain. Treatment has to be coordinated with the patient in terms of participative decisions.

Epidermal Rac1 regulates the DNA damage response and protects from UV-light-induced keratinocyte apoptosis and skin carcinogenesis.

Non-melanoma skin cancer (NMSC) is the most common type of cancer. Increased expression and activity of Rac1, a small Rho GTPase, has been shown previously in NMSC and other human cancers; suggesting that Rac1 may function as an oncogene in skin. DMBA/TPA skin carcinogenesis studies in mice have shown that Rac1 is required for chemically induced skin papilloma formation. However, UVB radiation by the sun, which causes DNA damage, is the most relevant cause for NMSC. A potential role of Rac1 in UV-light-induced skin carcinogenesis has not been investigated so far. To investigate this, we irradiated mice with epidermal Rac1 deficiency (Rac1-EKO) and their controls using a well-established protocol for long-term UV-irradiation. Most of the Rac1-EKO mice developed severe skin erosions upon long-term UV-irradiation, unlike their controls. These skin erosions in Rac1-EKO mice healed subsequently. Surprisingly, we observed development of squamous cell carcinomas (SCCs) within the UV-irradiation fields. This shows that the presence of Rac1 in the epidermis protects from UV-light-induced skin carcinogenesis. Short-term UV-irradiation experiments revealed increased UV-light-induced apoptosis of Rac1-deficient epidermal keratinocytes in vitro as well as in vivo. Further investigations using cyclobutane pyrimidine dimer photolyase transgenic mice revealed that the observed increase in UV-light-induced keratinocyte apoptosis in Rac1-EKO mice is DNA damage dependent and correlates with caspase-8 activation. Furthermore, Rac1-deficient keratinocytes showed reduced levels of p53, γ-H2AX and p-Chk1 suggesting an attenuated DNA damage response upon UV-irradiation. Taken together, our data provide direct evidence for a protective role of Rac1 in UV-light-induced skin carcinogenesis and keratinocyte apoptosis probably through regulating mechanisms of the DNA damage response and repair pathways.

Deletion of epidermal Rac1 inhibits HPV-8 induced skin papilloma formation and facilitates HPV-8- and UV-light induced skin carcinogenesis.

Overexpression and increased activity of the small Rho GTPase Rac1 has been linked to squamous cell carcinoma of the epidermis and mucosa in humans. Targeted deletion of Rac1 or inhibition of Rac1 activity in epidermal keratinocytes reduced papilloma formation in a chemical skin carcinogenesis mouse model. However, a potential role of Rac1 in HPV- and UV-light induced skin carcinogenesis has not been investigated so far, solar UV radiation being an important carcinogen to the skin.To investigate this, we deleted Rac1 or modulated its activity in mice with transgenic expression of Human papilloma virus type-8 (HPV-8) in epidermal keratinocytes. Our data show that inhibition or deletion of Rac1 results in reduced papilloma formation upon UV-irradiation with a single dose, whereas constitutive activation of Rac1 strongly increases papilloma frequency in these mice. Surprisingly, we observed that, upon chronic UV-irradiation, the majority of mice with transgenic expression of HPV-8 and epidermis specific Rac1 deletion developed squamous cell carcinomas. Taken together, our data show that Rac1 exerts a dual role in skin carcinogenesis: its activation is, on one hand, required for HPV-8- and UV-light induced papilloma formation but, on the other, suppresses the development of squamous cell carcinomas.

[Anchor-based ascertaining of meaningful changes in depressive symptoms using the example of the German short form of the CES-D]. / Ankerbasierte Ermittlung klinisch relevanter Veränderung depressiver Symptomatik am Beispiel der Kurzform der CES-D.

BACKGROUND AND OBJECTIVES: Depression is frequently measured on the 15 item version of the Center for Epidemiological Studies Depression Scale (CES-D-15). Up to now, there are no data based estimates for minimal clinically important differences (MCID) in depression scales. METHODS: Data on 5241 subjects with depressive symptoms from a German psychosomatic hospital were used. The changes in the CES-D-15 from admission to discharge were compared to the clinically global impression of change (CGIC), i. e., the therapists' perception. Categories of "slightly improved" and "much improved" were used as indicators of a clinically important difference. The relation between these ratings and the CES-D-15 was explored using mean change scores and sensitivity/specificity analyses. RESULTS: On average, a reduction of approximately nine points or 30 percent in the CES-D-15 or an individual effect size of 0.9 represented a minimal clinically important difference. A consistent relationship between the changes in the CES-D-15 and the CGIC was demonstrated irrespective of age, gender, education level, type of treatment, or first diagnosis. However, higher baseline scores in CES-D-15 required larger changes of raw values to represent a clinically important difference. CONCLUSIONS: Our results show anchor-based values for change in CES-D-15 that best represent a minimal clinically important difference. Reporting the percentage of patients who have reached the MCID improves the evaluation of therapeutical processes. The estimated MCID could also be used as an indicator for relevant changes in clinical practice. A further examination in other patient populations will be needed.

Using ICT to Support Individual Guidance in Health Promotion Programs for Increased Physical Activity.

INTRODUCTION: We report on experiences in implementing a system to support the individual guidance of training in health promotion programs aiming to increase participants' regular level of physical activity. METHODS: We used an iterative development approach considering data privacy and security aspects, followed by a phase of field testing and continuous further development. RESULTS: Our preliminary results comprise identified clinically relevant parameters, suitable data collection methods, experienced privacy and security challenges and a glance on our developed prototype system. DISCUSSION: We consider our results to be of interest for others doing related research. The most important requirements for a simple supporting system can be fulfilled with established solutions in the short run. A more adaptable and flexible system with an increased level of support in analysing the data, which we aim to achieve, leads to currently open research challenges.

Epidermal RelA specifically restricts contact allergen-induced inflammation and apoptosis in skin.

Strong inhibition of NF-κB signaling in the epidermis results in spontaneous skin inflammation in mice and men. As there is evidence for linkage between polymorphisms within the NF-κB signaling pathway and human inflammatory skin phenotypes, we asked whether partial functional inhibition of NF-κB signaling in epidermal keratinocytes can modulate clinically relevant skin inflammation. We therefore mutated rela specifically in the epidermis of mice (RelA(E-MUT) mice). These mice show no inflammatory phenotype. Induction of contact allergy, but not croton oil-induced irritant dermatitis, resulted in stronger ear swelling and increased epidermal thickness in RelA(E-MUT) mice. Both contact allergen and croton oil treatment led to increased expression of calgranulins A and B (S100A8/A9) in RelA(E-MUT) mice. Epidermal hyperproliferation in RelA(E-MUT) mice was non-cell autonomous as cultured primary epidermal keratinocytes from RelA(E-MUT) mice showed reduced proliferation compared with controls. These results demonstrate that epidermal RelA specifically regulates delayed-type hypersensitivity-induced skin inflammation. In addition, we describe here an essential but nonspecific function of RelA in the protection of epidermal keratinocytes from apoptosis. Our study identifies functions of NF-κB signaling in the epidermis and corroborates a specific role of epidermal keratinocytes in the regulation of skin inflammation.

Mechanisms regulating skin immunity and inflammation.

Immune responses in the skin are important for host defence against pathogenic microorganisms. However, dysregulated immune reactions can cause chronic inflammatory skin diseases. Extensive crosstalk between the different cellular and microbial components of the skin regulates local immune responses to ensure efficient host defence, to maintain and restore homeostasis, and to prevent chronic disease. In this Review, we discuss recent findings that highlight the complex regulatory networks that control skin immunity, and we provide new paradigms for the mechanisms that regulate skin immune responses in host defence and in chronic inflammation.

Tumor necrosis factor receptor signaling in keratinocytes triggers interleukin-24-dependent psoriasis-like skin inflammation in mice.

Psoriasis is a common chronic inflammatory skin disease with a prevalence of about 2% in the Caucasian population. Tumor necrosis factor (TNF) plays an essential role in the pathogenesis of psoriasis, but its mechanism of action remains poorly understood. Here we report that the development of psoriasis-like skin inflammation in mice with epidermis-specific inhibition of the transcription factor NF-κB was triggered by TNF receptor 1 (TNFR1)-dependent upregulation of interleukin-24 (IL-24) and activation of signal transducer and activator of transcription 3 (STAT3) signaling in keratinocytes. IL-24 was strongly expressed in human psoriatic epidermis, and pharmacological inhibition of NF-κB increased IL-24 expression in TNF-stimulated human primary keratinocytes, suggesting that this mechanism is relevant for human psoriasis. Therefore, our results expand current views on psoriasis pathogenesis by revealing a new keratinocyte-intrinsic mechanism that links TNFR1, NF-κB, ERK, IL-24, IL-22R1, and STAT3 signaling to disease initiation.

CCR2 recruits an inflammatory macrophage subpopulation critical for angiogenesis in tissue repair.

Monocytes/macrophages are critical in orchestrating the tissue-repair response. However, the mechanisms that govern macrophage regenerative activities during the sequential phases of repair are largely unknown. In the present study, we examined the dynamics and functions of diverse monocyte/macrophage phenotypes during the sequential stages of skin repair. By combining the analysis of a new CCR2-eGFP reporter mouse model with conditional mouse mutants defective in myeloid cell-restricted CCR2 signaling or VEGF-A synthesis, we show herein that among the large number of inflammatory CCR2(+)Ly6C(+) macrophages that dominate the early stage of repair, only a small fraction strongly expresses VEGF-A that has nonredundant functions for the induction of vascular sprouts. The switch of macrophage-derived VEGF-A during the early stage of tissue growth toward epidermal-derived VEGF-A during the late stage of tissue maturation was critical to achieving physiologic tissue vascularization and healing progression. The results of the present study provide new mechanistic insights into CCR2-mediated recruitment of blood monocyte subsets into damaged tissue, the dynamics and functional consequences of macrophage plasticity during the sequential repair phases, and the complementary role of macrophage-derived VEGF-A in coordinating effective tissue growth and vascularization in the context of tissue-resident wound cells. Our findings may be relevant for novel monocyte-based therapies to promote tissue vascularization.

A function for Rac1 in the terminal differentiation and pigmentation of hair.

The small GTPase Rac1 is ubiquitously expressed in proliferating and differentiating layers of the epidermis and hair follicles. Previously, Rac1 was shown to regulate stem cell behaviour in these compartments. We have asked whether Rac1 has, in addition, a specific, stem-cell-independent function in the regulation of terminal hair follicle differentiation. To address this, we have expressed a constitutively active mutant of Rac1, L61Rac1, only in the basal epidermal layer and outer root sheath of mice possessing an epidermis-specific deletion of endogenous Rac1, which experience severe hair loss. The resulting 'rescue' mice exhibited a hair coat throughout their lives. Therefore, expression of Rac1 activity in the keratin-14-positive compartment of the skin is sufficient for the formation of hair follicles and hair in normal quantities. The quality of hair formed in rescue mice was, however, not normal. Rescue mice showed a grey, dull hair coat, whereas that of wild-type and L61Rac1-transgenic mice was black and shiny. Hair analysis in rescue mice revealed altered structures of the hair shaft and the cuticle and disturbed organization of medulla cells and pigment distribution. Disorganization of medulla cells correlates with the absence of cortical, keratin-filled spikes that normally protrude from the cortex into the medulla. The desmosomal cadherin Dsc2, which normally decorates these protrusions, was found to be reduced or absent in the hair of rescue mice. Our study demonstrates regulatory functions for Rac1 in the formation of hair structure and pigmentation and thereby identifies, for the first time, a role for Rac1 in terminal differentiation.

The adaptor protein FADD protects epidermal keratinocytes from necroptosis in vivo and prevents skin inflammation.

Epidermal keratinocytes provide an essential structural and immunological barrier forming the first line of defense against potentially pathogenic microorganisms. Mechanisms regulating barrier integrity and innate immune responses in the epidermis are important for the maintenance of skin immune homeostasis and the pathogenesis of inflammatory skin diseases. Here, we show that epidermal keratinocyte-restricted deficiency of the adaptor protein FADD (FADD(E-KO)) induced severe inflammatory skin lesions in mice. The development of skin inflammation in FADD(E-KO) mice was triggered by RIP kinase 3 (RIP3)-mediated programmed necrosis (termed necroptosis) of FADD-deficient keratinocytes, which was partly dependent on the deubiquitinating enzyme CYLD and tumor necrosis factor (TNF)-TNF receptor 1 signaling. Collectively, our findings provide an in vivo experimental paradigm that regulation of necroptosis in keratinocytes is important for the maintenance of immune homeostasis and the prevention of chronic inflammation in the skin.

An open-label pilot study of the efficacy and safety of anakinra in patients with psoriatic arthritis refractory to or intolerant of methotrexate (MTX).

Only limited data have been published about the therapeutic use of anakinra in patients with psoriatic arthritis. We undertook this study to evaluate the efficacy and safety of anakinra in patients with active psoriatic arthritis. In a prospective open-label single-center study, 20 patients were treated with 100 mg anakinra everyday either alone or in combination with ongoing methotrexate over 6 months. Safety and efficacy was evaluated using Psoriasis Arthritis Response Criteria (PsARC), Disease Activity Score (DAS) 28, American College of Rheumatology (ACR), European League Against Rheumatism (EULAR), Psoriasis Area and Severity Index Score, Dactylitis Score and Health Assessment Questionnaire (HAQ), and the C-reactive protein, and erythrocyte sedimentation rate. Of the 20 patients enrolled, six completed 24 weeks, 18 completed 12 weeks, and 19 completed 4 weeks of treatment. Early-treatment termination was mainly due to inefficacy (13 patients) and only one drop-out occurred because of an unrelated adverse event. Six patients fulfilled continuously the PsARC until week 24. A moderate EULAR response was achieved by four patients and a good EULAR response by three patients in week 24. Five patients reached ACR 20, four patients ACR 50, and two patients ACR 70 in week 24. HAQ improved slightly throughout the study (n = 19, mean (SD); baseline, 1.127 (0.671); week 24, 1.055 (0.812)) just as DAS 28 (n = 16; baseline, 4.7(1.5); week 24, 4.0(2.0)). Only nine patients showed skin manifestations affecting >3% of their body surface area which improved in two, worsened in four, stabilized in two patients, and newly evolved in one patient. Adverse events were mainly mild (95%). Fifteen (75%) patients showed injection site reactions. No serious infections occurred. Anakinra was well tolerated with no occurrence of serious drug-associated adverse events and lead to improvement of signs and symptoms in nine out of 19 patients, therefore providing a potential therapeutic option in patients with active psoriatic arthritis.

Establishment of spontaneously immortalized keratinocyte lines from wild-type and mutant mice.

A considerable number of transgenic or knockout mice in which epidermal keratinocytes have been targeted die shortly after birth due to barrier defects. In this case, recovery and cultivation of keratinocytes from these animals provide an opportunity for in vitro studies. Working with isolated keratinocytes is also interesting for certain experiments which cannot be performed in live animals. Primary human keratinocytes can be kept in culture for a variable number of passages and then senescence. Immortalization can be achieved by transduction with constructs encoding viral genes. Murine keratinocytes can be kept in culture as primary cells. Naturally the numbers of cells obtained by direct isolation from mouse epidermis is restricted and sometimes not sufficient for certain biochemical analyses. To overcome this restriction some permanent murine keratinocyte lines have been generated by transfection with SV40T or HPV E6E7 genes. This is, however, not suitable if established or hypothetical biochemical links exist between these genes and the pathways or processes to be analysed in the respective experiment. We describe an easy and reproducible method of establishing permanent keratinocyte lines from spontaneously immortalized primary murine keratinocytes. This method employs co-cultivation of keratinocytes with 3T3-J2 fibroblast feeder cells for several passages during which immortalization occurs. The resulting keratinocyte lines do not only grow infinitely but, in many cases, individual lines from the same genetic background also exhibit similar growth characteristics, hence they are especially valuable for comparative studies.

ALX4 dysfunction disrupts craniofacial and epidermal development.

Genetic control of craniofacial morphogenesis requires a complex interaction of numerous genes encoding factors essential for patterning and differentiation. We present two Turkish families with a new autosomal recessive frontofacial dysostosis syndrome characterized by total alopecia, a large skull defect, coronal craniosynostosis, hypertelorism, severely depressed nasal bridge and ridge, bifid nasal tip, hypogonadism, callosal body agenesis and mental retardation. Using homozygosity mapping, we mapped the entity to chromosome 11p11.2-q12.3 and subsequently identified a homozygous c.793C-->T nonsense mutation in the human ortholog of the mouse aristaless-like homeobox 4 (ALX4) gene. This mutation is predicted to result in a premature stop codon (p.R265X) of ALX4 truncating 146 amino acids of the protein including a part of the highly conserved homeodomain and the C-terminal paired tail domain. Although the RNA is stable and not degraded by nonsense-mediated RNA decay, the mutant protein is likely to be non-functional. In a skin biopsy of an affected individual, we observed a hypomorphic interfollicular epidermis with reduced suprabasal layers associated with impaired interfollicular epidermal differentiation. Hair follicle-like structures were present but showed altered differentiation. Our data indicate that ALX4 plays a critical role both in craniofacial development as in skin and hair follicle development in human.