Klippel‐Feil syndrome in a boy exposed inadvertently to cyclophosphamide during pregnancy: A case report

BACKGROUND Cyclophosphamide (CPA) is an alkylating agent widely used as an immunosuppressive agent in the treatment of several autoimmune diseases, including systemic lupus erythematosus. Its teratogenic effect has been well studied in different experimental mammalian and non‐mammalian animal models. In humans, 11 cases of CPA teratogenesis have been documented. CASE We present a case of a patient with Klippel‐Feil syndrome inadvertently exposed to CPA and prednisone in utero during the first trimester. CONCLUSIONS This case of possible cyclophosphamide embryopathy provides evidence of teratogenesis as an etiologic agent in developmental field defects such as Klippel‐Feil syndrome. Birth Defects Research (Part A) 2012. © 2012 Wiley Periodicals, Inc.     

Report of a girl with Klippel-feil syndrome and Poland anomaly

Report of a girl with Klippel-feil syndrome and Poland anomaly: Klippel-Feil syndrome, consisting of the triad of a short neck, low posterior hairline, and limitation of neck movement, is a congenital anomaly characterized by the fusion of cervical vertebrae, Poland anomaly consists of unilateral aplasia of the chest wall muscles and ipsilateral anomalies of upper extremity. We report a 7-year-old girl with typical findings of Klippel-Feil syndrome and Poland anomaly. To the best of our knowledge a case of Klippel-Feil syndrome and Poland anomaly has not been described before, although a combination of Poland, Klippel-Feil and Moebius anomalies has been reported in the literature.     

Klippel-Feil syndrome and Dandy-Walker malformation

The Klippel-Feil deformity is a complex of osseous and visceral anomalies, which include low hairline, platybasia, fused cervical vertebrae with a short neck, and deafness. Associated central nervous system abnormalities include occipital cephalocele, Chiari I malformation, syrinx, microcephaly, and hydrocephalus. Herein, we report a case with Klippel-Feil syndrome and Dandy-Walker malformation.     

Antiandrogens: clinical applications

Antiandrogens, preventing androgen action at target tissue level, are used in the treatment of various androgen-dependent diseases. Pharmacologically these substances have either a steroidal structure, like cyproterone acetate (CPA) and spironolactone (SPL), or a non-steroidal structure, like flutamide (FLU). In women with hyperandrogenism (PCO syndrome, idiopathic hirsutism, acne), clinical benefit may be obtained with CPA, which also displays a progestational activity and an antigonadotropic effect. CPA (25-50 mg/day) is used in combination with ethinyl-estradiol (EE) (20-30 micrograms/day) in reversed sequential regimen. SPL, less effective than CPA may be employed in moderate hirsutism and acne at dosages of 100-200 mg/day. During SPL treatment menstrual irregularities are frequent: in this case an association with oral contraceptives is indicated. SPL + bromocriptine (2.5-5 mg/day) has been experienced with success in PCO syndrome. The pure antiandrogen FLU, inducing progressive increase in LH and testosterone secretion, may be used only in combination with oral contraceptives. In men antiandrogens have been tested in BPH and prostatic carcinoma. In BPH the decrease in nuclear receptors and DHT nuclear content during CPA or FLU may represent the rational base of the medical treatment. An improvement in urinary obstructive manifestation has been observed with CPA alone or associated with tamoxifen (100 mg + 100 mg day). In advanced prostatic carcinoma antiandrogens represent a good alternative to estrogen therapy with less side effects and in combination with surgical or medical castration (LH-RH analogues) achieve a complete androgen blockade. An increase in the percentage of remissions and survival has been reported.     

Preliminary report of novel technique for cryopreservation--vacuum-assisted cryoprotectant infiltration

To date, cryopreservation of large soft tissues has not been successfully achieved because of limitation of cryoprotective agent (CPA) infiltration into the tissue. This study aimed to investigate the effects of a vacuum on the tissue-infiltration of a CPA. An instant pickle-maker was modified for use as a vacuum apparatus, and glycerol was selected as the CPA. Twenty-six rats were used, and their thighs were divided into three treatment groups. Group 1: fresh control; Group 2: cryopreserved control, i.e., immersed in the CPA for 1h under atmospheric pressure and cryopreserved; Group 3: vacuum-assisted CPA infiltration, i.e., immersed in the CPA under negative pressure (20, 40 and 60 cmHg, for durations of 10, 20 and 30 min at each) and cryopreserved. The Groups 2 and 3 specimens were thawed after 3 weeks of cryopreservation at -80 °C and histologically examined, in comparison with Group 1. Skin: in Groups 2 and 3, the skin was well preserved. Muscle: in Group 2, both extracellular and intracellular ice crystal formation was widely distributed throughout the muscle tissue. In Group 3, under an adequate vacuum, the muscle tissue was well preserved, with no ice crystal formation. However, when the treatment was conducted under excessive vacuum conditions, the muscle tissue showed focal necrosis. Blood vessels: in Group 3, both the arteries and veins were well preserved up to the tunica intima. The method described in this paper may be a useful technique for achieving cryopreservation of large soft tissues.     

Carboxypeptidase A6 in zebrafish development and implications for VIth cranial nerve pathfinding

Carboxypeptidase A6 (CPA6) is an extracellular protease that cleaves carboxy-terminal hydrophobic amino acids and has been implicated in the defective innervation of the lateral rectus muscle by the VIth cranial nerve in Duane syndrome. In order to investigate the role of CPA6 in development, in particular its potential role in axon guidance, the zebrafish ortholog was identified and cloned. Zebrafish CPA6 was secreted and interacted with the extracellular matrix where it had a neutral pH optimum and specificity for C-terminal hydrophobic amino acids. Transient mRNA expression was found in newly formed somites, pectoral fin buds, the stomodeum and a conspicuous condensation posterior to the eye. Markers showed this tissue was not myogenic in nature. Rather, the CPA6 localization overlapped with a chondrogenic site which subsequently forms the walls of a myodome surrounding the lateral rectus muscle. No other zebrafish CPA gene exhibited a similar expression profile. Morpholino-mediated knockdown of CPA6 combined with retrograde labeling and horizontal eye movement analyses demonstrated that deficiency of CPA6 alone did not affect either VIth nerve development or function in the zebrafish. We suggest that mutations in other genes and/or enhancer elements, together with defective CPA6 expression, may be required for altered VIth nerve pathfinding. If mutations in CPA6 contribute to Duane syndrome, our results also suggest that Duane syndrome can be a chondrogenic rather than a myogenic or neurogenic developmental disorder.     

Substrate Specificity of Human Carboxypeptidase A6

Carboxypeptidase A6 (CPA6) is an extracellular matrix-bound metallocarboxypeptidase (CP) that has been implicated in Duane syndrome, a neurodevelopmental disorder in which the lateral rectus extraocular muscle is not properly innervated. Consistent with a role in Duane syndrome, CPA6 is expressed in a number of chondrocytic and nervous tissues during embryogenesis. To better characterize the enzymatic function and specificity of CPA6 and to compare this with other CPs, CPA6 was expressed in HEK293 cells and purified. Kinetic parameters were determined using a panel of synthetic carboxypeptidase substrates, indicating a preference of CPA6 for large hydrophobic C-terminal amino acids and only very weak activity toward small amino acids and histidine. A quantitative peptidomics approach using a mixture of peptides representative of the neuropeptidome allowed the characterization of CPA6 preferences at the P1 substrate position and suggested that small and acidic P1 residues significantly inhibit CPA6 cleavage. Finally, a comparison of available kinetic data for CPA enzymes shows a gradient of specificity across the subfamily, from the very restricted specificity of CPA2 to the very broad activity of CPA4. Structural data and modeling for all CPA/B subfamily members suggests the structural basis for the unique specificities observed for each member of the CPA/B subfamily of metallocarboxypeptidases.     

The association of spinal retroflexion with limb anomalies

The spectrum of conditions in which retroflexion of the spine occurs includes anencephaly, iniencephaly, and Klippel-Feil syndrome. It was suggested by Gimour ('41) that a continuum may exist linking the latter two entities. The present paper attempts to reopen this question by reporting two specimens which have none of the above conditions, yet have spinal retroflexion and limb malformations. Combined with other isolated case reports, they show that spinal retroflexion can arise over a variable period of time.     

Transmission Characteristics of the Clover Phyllody Agent by Dodder*

The results obtained in investigations on the biological characteristics of the clover phyllody agent (CPA) in respect to its transmission by the experimental vector Cuscuta campestris Younk, using Catbarantus roseus L. as constant host, are reported. Transmission efficiency of CPA was comparable whether the “stable bridge” or the “cut strand” of dodder method was adopted. The acquisition and inoculation threshold was between 4 and 6 days. The efficiency of transmission became greater by lengthening the test period up to 15 days. In cuttings of dodder maintained in water after recision, the maximum retention of infectivity, in respect to transmission capacity of CPA, was 28 days. It was proved that CPA invades C. campestris in a persistent manner. In comparative tests between CPA and APA (apple proliferation agent) it was demonstrated that the former is more efficiently transmitted than the latter. Moreover there is a much higher pathogenic effect of APA than CPA in dodder; in fact C. campestris as well as C. subinclusa Dur. and Hilg. develops poorly and shows deformations when growing on AP infected periwinkles. The basic distinguishable symptoms of the two diseases in C. roseus are: virescence and phyllody for CP; small but never virescent flowers for AP. MLOs have been detected, by electron microscopy, in sieve elements of C. roseus and C. campestris infected by CPA.     

Enzyme immobilization via monoclonal antibodies I. Preparation of a highly active immobilized carboxypeptidase A

A novel method for the preparation of highly active immobilized enzymes is described. It is based on the binding of enzymes to suitable carriers via monoclonal antibodies, which bind to the enzyme with high affinity without affecting its catalytic activity. The applicability of the method forwarded has been illustrated by the preparation of two samples of highly active immobilized carboxypeptidase A (CPA) preparations as follows: A mouse monoclonal antibody (mAb 100)to CPA that binds to the enzyme with a high-affinity constant without affecting its catalytic activity was prepared, purified, and characterized. Covalent binding of this monoclonal antibody to Eupergit C (EC) or noncovalent binding to Sepharose-protein A (SPA)yielded the conjugated carriers EC-mAb and SPA.mAb, respectively, which reacted specifically with CPA to give the immobilized enzyme preparations EC-mAb.CPA and SPA.mAb.CPA displaying full catalytic activity and improved stability. At pH 7.5 and a temperature range of 4-37 degrees C an apparent binding constant of approximately 10(8)M(-1) characterizing the interaction of CPA with EC-mAb and SPA.mAb, was obtained. To compare the properties of EC-mAb.CPA and SPA.mAb.CPA with those of immobilized CPA preparations obtained by some representative techniques of covalent binding of the enzyme with a corresponding carrier, the following immobilized CPA preparations were obtained and their properties investigated: EC-CPA (I), a preparation obtained by direct binding of EC with CPA; EC-NH-GA-CPA (II), a derivative obtained by covalent binding of CPA to aminated EC via glutaraldehyde; EC-NH-Su-CPA (III), a CPA derivative obtained by binding the enzyme to aminated EC via a succinyl residue; and EC-HMD-GA-CPA (IV), obtained by binding the enzyme via glutaraldehyde to a hexamethylene diamine derivative of EC. Full enzymic activity for all of the bound enzyme, such as that recorded for the immobilized CPA preparations EC-mAb.CPA and SPA.mAb.CPA, was not detected in any of the insoluble covalently bound enzyme preparations.     

The novel imprinted carboxypeptidase A4 gene ( CPA4) in the 7q32 imprinting domain

By a search for novel human imprinted genes in the vicinity of the imprinted gene MEST, at chromosome 7q32, we identified the carboxypeptidase A4 gene ( CPA4) in a gene cluster of the carboxypeptidase family, 200 kb centromeric to MEST. Because CPA4 was originally identified as a protein induced in a prostate cancer cell line (PC-3) by histone deacetylase inhibitors, and was located at the putative prostate cancer-aggressiveness locus at 7q32, we investigated its imprinting status in fetal tissues and in adult benign hypertrophic prostate (BPH). RT-PCR using four intragenic polymorphisms as markers showed that CPA4 was expressed preferentially from the maternal allele in the fetal heart, lung, liver, intestine, kidney, adrenal gland, and spleen, but not in the fetal brain. It was also preferentially expressed in the BPH. These findings support that CPA4 is imprinted and may become a strong candidate gene for prostate cancer-aggressiveness. As a Silver-Russell syndrome (SRS) locus has been proposed to be located to a region near MEST and to be involved in imprinting, CPA4 would have been a candidate gene for SRS. However, analysis of ten SRS patients revealed no mutations in CPA4.     

Mandibular herpes zoster; with report on the use of cortisone in a case with geniculate ganglion symptoms

Herpes zoster, an acute specific viral infection, occurs more commonly than is generally supposed. It should be differentiated from other diseases involving the ear and skin; it must be considered as a possible etiologic agent in some palsies of the facial, glossopharyngeal or vagal nerves. The type of cephalic herpes zoster should be carefully differentiated; cases involving the "geniculate zone" may be other than "Ramsay Hunt's syndrome." This syndrome is now defined as a herpes zoster eruption of the external ear at the "geniculate zone" with involvement of the seventh or seventh and eighth nerves. The "topognostic" method is the best for determining the level at which the facial nerve has been affected. It is questioned whether there is a single outstanding therapeutic agent for this disease. Cortisone had no apparent therapeutic effect in a case reported herein.     

Femoral hypoplasia-unusual facies syndrome in a black African infant

The femoral hypoplasia-unusual facies syndrome is a very rare association of femora and facial abnormalities. The most common features include hypoplasia of the femora and a characteristic facies with a short nose, long philtrum, thin upper lip and micrognathia. Maternal diabetes mellitus has been mainly identified as the causal agent. We reported the first case in a black African and discuss prenatal diagnosis and aetiology.     

A spontaneously occuring mammary gland ductal carcinoma in situ in a rhesus macaque (Macaca mulatta) and a review of spontaneous mammary gland tumors in rhesus monkeys

A spontaneous mammary gland ductal carcinoma in situ was diagnosed in a 6-8-year-old female rhesus macaque (Macaca mulatta). To our knowledge, this is only the tenth case of spontaneous mammary gland tumors to be reported in rhesus monkeys. Despite the paucity of case reports, several theories exist to explain the occurrence of mammary tumors. The Mason Pfizer monkey virus, a type D retrovirus similar to the virus that causes simian acquired immunodeficiency syndrome, has been implicated as a possible etiologic agent. Because this virus has been isolated from normal primate mammary tissue, it is unlikely to be the sole etiologic agent. Other theories include the tumorogenic effects that androgens, growth hormones, irradiation, and aging have on the mammary gland.     

An analytical model for the prediction of the local concentration of cryophylactic agents in perfused organs.

An analytical model has been developed for the prediction of the spatial and temporal distribution of the concentration of cryophylactic agent (CPA) in perfused tissues and organs. Perfusate concentration of the CPA is allowed to change according to a protocol in which the concentration increases linearly from zero during a time interval t₀. Between time t₀ and t_f, the concentration is constant.The numerical results of this analysis show that the typical time required to reach an appropriate CPA concentration by simple diffusion alone is sufficiently long that the natural vascular network must be employed as a means of introducing the CPA. The results of this analysis also demonstrate the importance of careful protocol design with the aid of this model. There is evidence to show that protocols previously reported in the literature failed to achieve the appropriate CPA concentration.Further work in this area requires more detailed information concerning the diffusion coefficients of the various CPA's in biological tissues. With the aid of this information and the present model, optimum protocols can be designed so that the CPA concentration can be increased to the desired level without inducing osmotic shock.     

Formulation development of the biocontrol agent Bacillus subtilis strain CPA-8 by spray-drying

Aims: To prepare commercially acceptable formulations of Bacillus subtilis CPA‐8 by spray‐drying with long storage life and retained efficacy to control peach and nectarine brown rot caused by Monilinia spp. Methods and Results: CPA‐8 24‐h‐ and 72‐h‐old cultures were spray dried using 10% skimmed milk, 10% skimmed milk plus 10% MgSO₄, 10% MgSO₄ and 20% MgSO₄ as carriers/protectants. All carriers/protectants gave good percentages of powder recovery (28–38%) and moisture content (7–13%). CPA‐8 survival varied considerably among spray‐dried 24‐h‐ and 72‐h‐old cultures. Seventy‐two hours culture spray dried formulations showed the highest survival (28–32%) with final concentration products of 1·6–3·3 × 10⁹ CFU g^?1, while viability of 24‐h‐old formulations was lower than 1%. Spray‐dried 72‐h‐old formulations were selected to subsequent evaluation. Rehydration of cells with water provided a good recovery of CPA‐8 dried cells, similar to other complex rehydration media tested. Spray‐dried formulations stored at 4 ± 1 and 20 ± 1°C showed good shelf life during 6 months, and viability was maintained or slightly decreased by 0·2–0·3‐log. CPA‐8 formulations after 4‐ and 6 months storage were effective in controlling brown rot caused by Monilinia spp. on nectarines and peaches resulting in a 90–100% reduction in disease incidence. Conclusions: Stable and effective formulations of biocontrol agent B. subtilis CPA‐8 could be obtained by spray‐drying. Significance and Impact of the Study: New shelf‐stable and effective formulations of a biocontrol agent have been obtained by spray‐drying to control brown rot on peach.     

Comparison of actual vs. synthesized ternary phase diagrams for solutes of cryobiological interest

Phase diagrams are of great utility in cryobiology, especially, those consisting of a cryoprotective agent (CPA) dissolved in a physiological salt solution. These ternary phase diagrams consist of plots of the freezing points of increasing concentrations of solutions of cryoprotective agents (CPA) plus NaCl. Because they are time-consuming to generate, ternary diagrams are only available for a small number of CPAs. We wanted to determine whether accurate ternary phase diagrams could be synthesized by adding together the freezing point depressions of binary solutions of CPA/water and NaCl/water which match the corresponding solute molality concentrations in the ternary solution. We begin with a low concentration of a solution of CPA+salt of given R (CPA/salt) weight ratio. Ice formation in that solution is mimicked by withdrawing water from it which increases the concentrations of both the CPA and the NaCl. We compute the individual solute concentrations, determine their freezing points from published binary phase diagrams, and sum the freezing points. These yield the synthesized ternary phase diagram for a solution of given R. They were compared with published experimental ternary phase diagrams for glycerol, dimethyl sulfoxide (DMSO), sucrose, and ethylene glycol (EG) plus NaCl in water. For the first three, the synthesized and experimental phase diagrams agreed closely, with some divergence occurring as wt% concentrations exceeded 30% for DMSO and 55% for glycerol, and sucrose. However, in the case of EG there were substantial differences over nearly the entire range of concentrations which we attribute to systematic errors in the experimental EG data. New experimental EG work will be required to resolve this issue.     

Characterization of the differences in the cyclopiazonic acid binding mode to mammalian and P. Falciparum Ca2+ pumps: A computational study

Despite the investments in malaria research, an effective vaccine has not yet been developed and the causative parasites are becoming increasingly resistant to most of the available drugs. PfATP6, the sarco/endoplasmic reticulum Ca2+ pump (SERCA) of P. falciparum, has been recently genetically validated as a potential antimalarial target and cyclopiazonic acid (CPA) has been found to be a potent inhibitor of SERCAs in several organisms, including P. falciparum. In position 263, PfATP6 displays a leucine residue, whilst the corresponding position in the mammalian SERCA is occupied by a glutamic acid. The PfATP6 L263E mutation has been studied in relation to the artemisinin inhibitory effect on P. falciparum and recent studies have provided evidence that the parasite with this mutation is more susceptible to CPA. Here, we characterized, for the first time, the interaction of CPA with PfATP6 and its mammalian counterpart to understand similarities and differences in the mode of binding of the inhibitor to the two Ca2+ pumps. We found that, even though CPA does not directly interact with the residue in position 263, the presence of a hydrophobic residue in this position in PfATP6 rather than a negatively charged one, as in the mammalian SERCA, entails a conformational arrangement of the binding pocket which, in turn, determines a relaxation of CPA leading to a different binding mode of the compound. Our findings highlight differences between the plasmodial and human SERCA CPA‐binding pockets that may be exploited to design CPA derivatives more selective toward PfATP6. Proteins 2015; 83:564–574. © 2015 The Authors. Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.     

Cryopreservation of keratinocytes in a monolayer.

The cryopreservation of cells in tissues is one of the major challenges in current cryobiology, especially with regard to the progressively increasing field of tissue engineering. It is very questionable whether protocols which were developed for the cryopreservation of isolated cells are also applicable for cells in more complex structures, such as tissues. As a starting point toward cryopreservation of these three-dimensional structures, the aim of this study was to find an optimum cryopreservation protocol for keratinocytes in a monolayer (two-dimensional structure). These epidermal cells can be transplanted as a monolayer grown on an appropriate matrix for the treatment of deep-dermal burns and leg ulcers. The successful cryopreservation of such transplants would offer the advantage of long-term storage and immediate availability of the transplant. In our study, the variables investigated were the cryoprotective solution and the cooling rate. In order to find a nontoxic cryoprotective agent (CPA) which could be transplanted without an additional washing step, we included hydroxyethyl starch (HES) as a possible CPA in our experimental protocol with the commonly used CPAs Me(2)SO, glycerol, and ethylene glycol. For the evaluation, the cell survival rate was determined by dye exclusion (trypan blue) and the cell metabolism was investigated by cell activity assay (alamarBlue). In conclusion, the cryopreservation protocol with 10 wt.-% HES resulted not only in the highest survival rate (72%) but also in the highest metabolic activity of the cells after thawing; comparable values for the other CPAs were: Me(2)SO, 48%; glycerol, 8%; and ethylene glycol, 10%.